Gas-fed fermentation reactors, systems and processes

ABSTRACT

Reactors, systems and processes for the production of biomass by culturing microorganisms in aqueous liquid culture medium circulating inner loop reactor which utilize nonvertical pressure reduction zones are described. Recovery and processing of the culture microorganisms to obtain products, such as proteins or hydrocarbons is described.

BACKGROUND Technical Field

This invention is related to reactors, systems and processes useful infermentation and, in particular, fermentation systems using a gaseoussubstrate.

Description of the Related Art

With the ever increasing depletion of fossil fuel deposits, theincreasing production of greenhouse gases and recent concerns aboutclimate change, substituting biofuels (e.g., ethanol, biodiesel) forfossil fuels has become an industrial focus. However, biofuels generatedto date have their own difficulties and concerns. First generationbiofuels are derived from plants (e.g., starch; cane sugar; and corn,rapeseed, soybean, palm, and other vegetable oils), but these fuel cropscompete with crops grown for human and animal consumption. The amount ofglobally available farm land is insufficient to satisfy the increasingneeds for both food and fuel. To reduce the demand placed upon foodproducers for biofuel compatible grains, second generation biofuelsusing alternative biological material such as cellulose or algae areunder development. However, technical difficulties in production, alongwith the high cost of production, have not made second generationbiofuels any more cost-effective or accessible.

Third or next generation biofuels are made using alternative, non-foodbased, carbon feedstocks. As part of this effort, the use ofalternative, non-biological based, feedstocks in the production ofhigher hydrocarbon compounds including fuels, lubricants, and plasticsis gaining ever-increasing momentum. Such feedstocks may include one ormore carbon-containing compounds or mixtures of carbon-containing andnon-carbon-containing compounds that include, among others, methane andsyngas. Methane, for example, is a relatively abundant, naturallyoccurring and found in many locations throughout the world. Methane isalso produced during many biological decay processes, and thus may becaptured from waste treatment and landfill facilities. For its relativeabundance, methane is a potent greenhouse gas, having 23× the relativegreenhouse gas contribution of CO₂. Historically, methane has beenviewed as a somewhat valuable byproduct that is difficult to convert tohigher value products or to transport to the marketplace from remote orstranded locations such as remote gas fields or off-shore productionplatforms. Methane from such sources, as well as the methane produced bybiological decomposition processes occurring at sewage treatmentfacilities and landfills, is primarily either vented or flared. Theability to economically and efficiently convert methane and similarcarbon-containing gases to one or more higher value C₂ or higherhydrocarbons would permit producers to take advantage of a relativelyabundant, non-biologically produced, feedstock while, at the same time,providing a significant environmental benefit.

The rise in domestic production of methane capability makes methane morereadily available domestically. Domestic natural gas is primarilyproduced by hydraulic fracturing (“fracking”), but methane can also beobtained from other sources, such as landfills and sewage. But methane'svolatility makes the transport and/or direct usage of methane as a fuelproblematic.

For these reasons, a strong incentive exists to convert the methane toone or more liquid products, for example motor fuels, to permit easiertransport to the point of use or sale. Two main approaches are currentlybeing pursued: liquefaction leading to liquefied natural gas (LNG) andchemical conversion to convert gas-to-liquid (GTL) (Patel, 2005, 7thWorld Congress of Chemical Engineering, Glasgow, Scotland, UK). TheFischer Tropsch (F-T) process is currently the most prevalent approachfor converting large quantities of methane to higher-order hydrocarbons(Patel, 2005). Note that the F-T process takes syngas as an input;syngas is produced from natural gas by steam reforming (syngas can alsobe sourced from coal gasification, by high temperature reaction withwater and oxygen). The F-T process yields petroleum products consistentwith today's fuel supply, but suffers from a number of drawbacks,including low yields, poor selectivity (making downstream utilizationcomplex), and requires significant capital expenditure and scale toachieve economical production (Spath and Dayton, December 2003NREL1TP-510-34929). The massive scale required for a F-T plant(generally in excess of two billion dollars in capital cost [Patel,2005]) also represents a significant limitation due to the large amountof methane feedstock required to offset the enormous capital cost of theF-T process. As methane transportation is prohibitively expensive inmost cases, such a plant must be co-located with a steady, reliable, andcost efficient source of methane, usually in the form of a significantmethane reservoir or a methane pipeline. An additional cost and scalingfactor is the economics of gas-scrubbing technologies (Spath and Dayton,2003), since F-T catalysts are quite sensitive to common contaminantsfound in natural gas that pass unaffected through the syngas conversionprocess.

The requirements for ready access to large volumes of a relatively cleanmethane-containing gas, combined with a massive capital investment,currently limit natural gas based F-T plants to successful andeconomically viable operation in only a few locations worldwide (Spathand Dayton, 2003). The high minimum processing requirement for agas-to-liquids process or liquefied natural gas plant, combined with thehigh cost of transport, result in smaller methane sources remaining as“stranded” gas deposits. Such stranded gas can include, but is notlimited to, natural gas produced at off-shore oil wells, or methaneoff-gas from landfills. Due to the current absence of efficientsmall-scale conversion technologies, such stranded gas sources aretypically vented to atmosphere or flared, as methane accumulationpresents a significant safety risk. Gas-to-liquids facilities using theFischer-Tropsch process have been in operation semi-continuously since1938. Several companies are currently investigating introduction of newplants given the current availability and price of methane discussedabove. However, despite significant research and development over thelast 70+ years, the limitations of Fischer-Tropsch technology preventbroad adoption of commercial gas-to-liquids processes.

Advances in the efficiency in animal feed utilization have been achievedover the past several decades through the use of feed additives. Theseadded substances augment the nutrient content, energy content, and/ordisease fighting properties of animal feed compositions. A growingchallenge for commercial animal producers is the rising cost of grain.The rising costs are due in part to competing demands for grains forbiofuel and human food use. With the rising cost of grain and proteincomplements, coupled with limited land available for feed production,alternative low-cost animal feed products with beneficial nutritive anddisease fighting properties are desirable.

A number of different protein-containing materials have been proposed assubstitutes for more traditional sources of protein, such as fish meal,soya products and blood plasma, in human foods and as animal feed. Theseprotein-containing materials include single cell microorganisms such asfungi, yeasts and bacteria which contain high proportions of proteins.These microorganisms may be grown on hydrocarbon or other substrates.

In view of the above, biological fermentation using C₁ substrates as acarbon source presents an attractive solution to both the currentcompetition between food sources and fermentation for producingchemicals/fuels, the need for alternative low-cost animal feed products,as well as the lack of good options for utilization of natural gas.However, fermentation of gaseous substrates such as methane, CO, or CO₂presents significant challenges due to the requirement that the carbonsubstrate must be transferred from the gas phase to an aqueous phase toallow for uptake and metabolism by the C₁ metabolizingnon-photosynthetic microorganisms in culture. Simultaneously, othergasses such as O₂ or H₂ may also be required to be transferred from thegas phase to allow cellular metabolism to progress (aerobic or anaerobicmetabolism, respectively). Waste products (such as CO₂ in the case ofaerobic metabolism) must be isolated from the microorganisms to allowfor efficient microbial growth. Further, the heat generation frommetabolism of C₁ substrates is significant and the system requirescooling to maintain optimal conditions for microbial growth.

Convective mass transfer from the liquid phase to the vapor phase can bedescribed with a mass transfer coefficient. The flux is equal to theproduct of the mass transfer coefficient, the surface area, and theconcentration difference (Flux=k A ΔC).

The mass transfer coefficient is influenced by a variety of factorsincluding the size of the molecule to be transferred, its solubility inthe aqueous phase, and the size of the boundary layer between the phases(typically controlled in fermentation systems by mixing speed andturbulence). The surface area between the gas and liquid phases in mostfermentation systems is primarily limited by the bubble size of theinput gas. Bubble size can be controlled by introducing the gas throughsmall pores, as well as increasing shear forces to break apart bubblesand prevent coalescence. The concentration difference can be theconcentration difference across the gas phase boundary layer, theconcentration difference across the liquid phase boundary layer, theconcentration difference between the bulk vapor and the vapor whichwould be in equilibrium with the bulk liquid, or the concentrationdifference between the bulk liquid and the liquid which would be inequilibrium with the bulk vapor. In most fermentation systems, theconcentration difference is controlled by the pressure of the gas phase.

Conventional fermentation systems (bioreactors) achieve gas mixing byone of two methods: stirring or airlift. Stirred fermentors achievemixing by means of stirring blades generally placed centrally in asingle large fermentor. The stirrer blades generate turbulence and shearin the liquid while gas bubbles are introduced at the bottom of thefermentor, thus impeding the progress of the bubbles as they travel upthe fermentor and shearing the gas bubbles to reduce the tendency of thebubbles to coalesce within the fermentor. The advantage of this type offermentor is the fast, relatively homogeneous mixing and gas bubbledispersion that is possible due to the high speed of the mixing blades.However, this type of fermentor can be difficult to scale-up, as theenergy requirements to obtain the same rate of mixing and mass transportcan be prohibitive as the volume increases. Further, the vigorous mixingimplies a significant heating of the fermentation liquid, and the use ofa single large fermentor limits the surface area available for heatexchange cooling.

Airlift fermentors avoid mechanical stirrers by incorporating a flowpath for the liquid. Airlift fermentors have a downflow and an upflowsection which are interconnected at both ends; these sections can eitherbe separate units (referred to as a loop fermentor), or concentric(airlift fermentor). In airlift fermentors, gasses are supplied at thebottom of the upflow section through a bubble-generating apparatus. Thebubbles mix with the liquid, reducing the density of the liquid andcausing the gas-liquid mixture to rise through the upflow section. Therising mixture displaces liquid at the top of the reactor, which travelsdown the downflow section to replace the liquid at the bottom,establishing a circular flow in the fermentor. In order to obtain a longresidence time for the gas bubbles in the liquid, airlift fermentors aregenerally tall and have a limited transverse cross-sectional area. Thisimplies that the gas must be supplied at a relatively high pressure toovercome hydrostatic pressure formed by the column of liquid present inthe fermentor. In addition, the bubble size increases significantlythroughout the fermentor as the pressure decreases with height. Theincreasing bubble diameter proportionately reduces the rate of masstransfer between the gas bubbles and the liquid phase by reducing theratio of gas bubble area (proportionate to the square of the gas bubbleradius) to gas bubble volume (proportionate to the cube of the gasbubble radius) through which mass transfer may occur. Flow rates andshear forces in airlift fermentors are significantly lower than instirred tank fermentors, which also tend to increase bubble coalescenceand reduce the efficiency of cooling the fermentor. Finally, separationof the unused and waste gases from the mixture exiting the upflowportion of the fermentor prior to the return of the liquid to thedownflow section can be challenging.

Loop reactors are described in U.S. Pat. No. 7,575,163 and have beenproposed for fermenting microorganisms, e.g., for the generation ofbiomass or for the preparation of materials produced by microorganisms.FIG. 1 illustrates one loop reactor 1 including an effluent gas removalzone 2 which flows into a vertical downflow zone 3. Effluent gas removalzone 2 includes an outlet port 7 and an emergency vent 8. Verticaldownflow zone 3 includes a nutrient gas inlet 15. A propeller 10 poweredby motor 11 assists in circulation of a liquid culture medium throughthe loop reactor. Upstream of propeller 10 is an exit port 12 forremoving material from the loop reactor. Downstream of propeller 10 areammonia and mineral inlets 17 and 18. Liquid culture medium 9 passesthrough a plurality of static mixers 14 in a horizontal section 4 of theloop reactor. The horizontal section of the loop reactor also includes aplurality of nutrient gas inlets 13. Downstream of the last static mixer14, the loop reactor includes a vertical upflow section 5. The top endof vertical upflow section 5 fluidly communicates with a horizontaloutflow zone 6. Vertical upflow section 5 is provided with a nutrientgas inlet 16. Downstream of nutrient gas inlet 16 is a drive gas inlet19 through which a driving gas is delivered to the liquid culturemedium. The '163 patent describes the loop reactor illustrated in FIG. 1has a vertical drop between the gas-liquid surface at the end of theoutflow zone 6 and the centerline of the loop in the horizontal sectionthat is at least 10 meters.

BRIEF SUMMARY

In one aspect, the present disclosure describes systems, processes andapparatuses for efficient mass transfer of gaseous substrates formicrobial fermentation. Additionally, this disclosure describes systems,processes and apparatuses for fermenting gaseous carbon-containingfeedstocks using a culture primarily comprising a C₁ metabolizingnon-photosynthetic microorganism. In other aspects, this disclosuredescribes systems, processes and apparatuses for fermenting gaseousfeedstocks which include gaseous substrates, using other than C₁metabolizing non-photosynthetic microorganism(s). In yet another aspect,this disclosure describes scalable fermentor designs for allowing highflux gas-phase to liquid-phase mass transfer in addition to efficientheat exchange and waste gas removal. Systems and processes forfermentation that overcome disadvantages known in the art and providethe public with new processes and devices for the optimal production ofa variety of products are described.

Such fermentation systems may employ one or more species ofmicroorganism that are capable of metabolizing gaseous compounds; forexample, C₁ compounds. Such microorganisms include prokaryotes orbacteria, such as Methylomonas, Methylobacter, Methylococcus,Methylosinus, Methylocystis, Methylomicrobium, Methanomonas,Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium,Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter,Rhodopseudomonas, or Pseudomonas. In some instances, the C₁ metabolizingmicroorganisms may include methanotrophs, methylotrophs or combinationsthereof. Preferred methanotrophs include Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium,Methanomonas, or combinations thereof. Exemplary methanotrophs includeMethylomonas sp. 16a (ATCC PTA 2402), Methylosinus trichosporium (NRRLB-ll, 196), Methylosinus sporium (NRRL B-ll, 197), Methylocystis parvus(NRRL B-ll, 198), Methylomonas methanica (NRRL B-5 11,199), Methylomonasalbus (NRRL B-ll, 200), Methylobacter capsulatus (NRRL B-11,201),Methylobacterium organophilum (ATCC 27,886), Methylomonas sp. AJ-3670(FERM P-2400), Methylomicrobium alcaliphilum, Methylocella silvestris,Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylosinustrichosporium OB3b, Methylococcus capsulatus Bath, Methylomonas sp. 16a,Methylomicrobium alcaliphilum 20Z, or high growth variants thereof.Preferred methylotrophs include Methylobacterium extorquens,Methylobacterium radiotolerans, Methylobacterium populi,Methylobacterium chloromethanicum, Methylobacterium nodulans, orcombinations thereof.

Microorganisms capable of metabolizing C₁ compounds found in syngasinclude, but are not limited to Clostridium, Moorella, Pyrococcus,Eubacterium, Desulfobacterium, Carboxydothermus, Acetogenium,Acetobacterium, Acetoanaerobium, Butyribacterium, Peptostreptococcus, orcombinations thereof. Exemplary methylotrophs include Clostridiumautoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei,Clostridium carboxydivorans, Butyribacterium methylotrophicum,Clostridium woodii, Clostridium neopropanologen, or combinationsthereof. In some instances, C₁ metabolizing microorganisms areeukaryotes such as yeast, including Candida, Yarrowia, Hansenula,Pichia, Torulopsis, or Rhodotorula.

In other instances, the C₁ metabolizing non-photosynthetic microorganismis an obligate C₁ metabolizing non-photosynthetic microorganism, such asan obligate methanotroph, an obligate methylotroph, or combinationsthereof. In some instances, the C₁ metabolizing non-photosyntheticmicroorganism is a recombinant microorganism comprising a heterologouspolynucleotide encoding a fatty acid producing enzyme, a formaldehydeassimilation enzyme, or combinations thereof.

In addition or as alternatives to the above, the present disclosuredescribes the following embodiments. A first embodiment directed to asystem for stimulating production of biomass that includes a loopreactor which includes a gas/liquid separation vessel for separating amulti-phase mixture of a gas and a liquid culture medium into a gasphase and a liquid phase, the gas/liquid separation vessel including anoutlet and an inlet; a loop section including an inlet in fluidcommunication with the outlet of the gas/liquid separation vessel, anoutlet in fluid communication with the inlet of the gas/liquidseparation vessel and a loop section centerline; a first non-verticalpressure reduction zone including a first pressure reduction device, thefirst non-vertical pressure reduction zone located between the inlet ofthe loop section and the outlet of the loop section, a vertical distancebetween the loop section centerline at the inlet of the gas/liquidseparation vessel and loop section centerline at the inlet of the loopsection is less than 8 meters.

A second embodiment disclosed herein is directed to the first embodimentwherein the pressure reduction device is a valve or expansion joint.

A third embodiment disclosed herein is directed to the system of thefirst and second embodiments, including a second pressure reduction zonedownstream of the first non-vertical pressure reduction zone.

A fourth embodiment disclosed herein is directed to the first throughthird embodiments wherein the second pressure reduction zone is a secondnon-vertical pressure reduction zone.

A fifth embodiment disclosed herein is directed to the first throughfourth embodiments wherein the vertical distance between the loopsection centerline at the inlet of the gas/liquid separation vessel andthe loop section centerline at the inlet of the loop section is lessthan 6 meters.

A sixth embodiment disclosed herein is directed to the first throughfifth embodiment wherein the vertical distance between the loop sectioncenterline at the inlet of the gas/liquid separation vessel and the loopsection centerline at the inlet of the loop section is less than 5meters.

A seventh embodiment disclosed herein is directed to the first throughsixth embodiments wherein the loop reactor further includes a desorptiongas inlet, the desorption gas inlet located in a non-vertical portion ofthe loop section of the loop reactor.

An eighth embodiment disclosed herein is directed to the first throughseventh embodiments wherein the first vertical pressure reduction deviceis a device that reduces pressure without relying upon a change inhydrostatic pressure.

A ninth embodiment disclosed herein is directed to a process forstimulating production of biomass including flowing through a loopsection of a loop reactor, a multi-phase mixture of a gas and a liquidculture medium, the loop section including a loop section centerline;introducing nutrients into the multi-phase mixture; introducing methaneand oxygen into the multi-phase mixture; passing the multi-phase mixtureof a gas and a liquid culture medium through a first non-verticalpressure reduction zone of the loop reactor, the first non-verticalpressure reduction zone of the loop reactor including a first pressurereduction device; separating the multi-phase mixture of a gas and liquidculture medium into a gas phase and a liquid phase downstream of thefirst pressure reduction device; flowing the gas phase and the liquidphase separated from the multi-phase mixture of a gas and a liquidculture medium into a gas/liquid separation vessel at an inlet to thegas/liquid separation vessel, the inlet to the gas/liquid separationvessel including a centerline; and removing the liquid phase from anoutlet of the gas/liquid separation vessel and delivering the removedliquid phase to an inlet of the loop section, a vertical distancebetween the loop section centerline at the inlet of the loop section andthe centerline of the inlet to the gas/liquid separation vessel beingless than 8 meters.

A tenth embodiment described herein is directed to the ninth embodimentwherein passing the multi-phase mixture of a gas and a liquid culturemedium to a first non-vertical pressure reduction zone includes passingthe multi-phase mixture of a gas and a liquid culture medium through avalve, expansion joint, static mixer or piping elbow.

An eleventh embodiment described herein is directed to the ninth andtenth embodiments further including passing the multi-phase mixture of agas and a liquid culture medium through a second pressure reduction zonedownstream of the first non-vertical pressure reduction zone.

A twelfth embodiment described herein is directed to the ninth througheleventh embodiments wherein the vertical distance between the loopsection centerline at the inlet of the loop section and the centerlineof the inlet to the gas/liquid separation vessel is less than 6 meters.

A thirteenth embodiment described herein is directed to the ninththrough twelfth embodiments wherein the vertical distance between theloop section centerline at the inlet of the loop section and thecenterline of the inlet to the gas/liquid separation vessel is less than5 meters.

A fourteenth embodiment described herein is directed to the ninththrough thirteenth embodiments, further comprising introducing adesorption gas into a non-vertical portion of the loop section of theloop reactor.

A fifteenth embodiment described herein is directed to the ninth throughfourteenth embodiments, further comprising passing the multi-phasemixture of a gas and a liquid culture medium through a firstnon-vertical pressure reduction zone, and includes passing themulti-phase mixture of a gas and a liquid culture medium through adevice that reduces pressure without relying upon a change inhydrostatic pressure.

A sixteenth embodiment described herein is directed to a process forstimulating the production of biomass in a loop reactor includingpassing a multi-phase mixture of a gas and a liquid culture mediumthrough a first non-vertical pressure reduction zone of the loopreactor, the first non-vertical pressure reduction zone of the loopreactor including a first pressure reduction device; separating themulti-phase mixture of a gas and a liquid culture medium into a gasphase and a liquid phase downstream of the first pressure reductiondevice; passing the gas phase and the liquid phase separated from themulti-phase mixture of a gas and a liquid culture medium into agas/liquid separation vessel at an inlet to the gas/liquid separationvessel, the inlet to the gas/liquid separation vessel including acenterline; and removing a liquid phase from an outlet of the gas/liquidseparation vessel and delivering the removed liquid phase to an inlet ofa loop section of the loop reactor, a vertical distance between the loopsection centerline at the inlet of the loop section and the centerlineof the inlet to the gas/liquid separation vessel being less than 8meters.

A seventeenth embodiment described herein is directed to the sixteenthembodiment wherein the vertical distance between the loop sectioncenterline at the inlet of the loop section and the centerline of theinlet to the gas/liquid separation vessel is less than 6 meters.

An eighteenth embodiment described herein is directed to the sixteenththrough seventeenth embodiments wherein the vertical distance betweenthe loop section centerline at the inlet of the loop section and thecenterline of the inlet to the gas/liquid separation vessel is less than5 meters.

A nineteenth embodiment described herein is directed to the sixteenththrough eighteenth embodiments wherein the first pressure reductiondevice is a device that reduces pressure without relying upon a changein hydrostatic pressure.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the drawings, the sizes and relative positions of elements in thedrawings are not necessarily drawn to scale. For example, the variouselements and angles are not drawn to scale, and some of these elementsare arbitrarily enlarged and positioned to improve drawing legibility.Further, the particular shapes of the elements as drawn are not intendedto convey any information regarding the actual shape of the particularelements, and have been selected solely for ease of recognition in thedrawings.

FIG. 1 shows a schematic view of a prior art loop reactor including avertical upflow section upstream from an outflow zone where degassingoccurs.

FIG. 2 shows a schematic block diagram of an example of a loop reactorfor stimulating production of biomass and optional subsystems accordingto one or more illustrated and described embodiments.

FIG. 3 shows a schematic view of an example system for stimulatingproduction of biomass that is useful in fermenting a gaseous substratethat includes a first pressure reduction zone and a second pressurereduction zone according to one or more illustrated and/or describedembodiments.

FIG. 4 shows a schematic view of an example system for stimulatingproduction of biomass that is useful in fermenting a gaseous substratethat includes a first pressure reduction zone according to one or moreillustrated and/or described embodiments.

FIG. 5 shows a high level flow diagram of a fermentation process thatincludes flowing a multi-phase mixture through a first pressurereduction zone of a loop reactor, according to one or more illustratedand/or described embodiments.

FIG. 6 shows a high level flow diagram of a fermentation process thatincludes flowing a multi-phase mixture through a first pressurereduction zone and a second pressure reduction zone of a loop reactor,according to one or more illustrated and/or described embodiments.

FIG. 7A is an elevational view a portion of a non-vertical pressurereduction device in accordance with one or more illustrated and/ordescribed embodiments.

FIG. 7B is an elevational view of a portion of a non-vertical pressurereduction device in accordance with one or more illustrated and/ordescribed embodiments.

FIG. 7C is an elevational view of a portion of a non-vertical pressurereduction device in accordance with one or more illustrated and/ordescribed embodiments.

FIG. 7D is an elevational view of a non-vertical pressure reductiondevice formed by assembling the portions of a non-vertical pressurereduction device illustrated in FIGS. 7A, 7B and 7C.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments.However, one skilled in the art will understand that the invention maybe practiced without these details. In other instances, structures,standard vessel design details, detailed design parameters of availablecomponents such as liquid or gas distributors, pumps, turbines, andsimilar, details concerning the design and construction of AmericanSociety of Mechanical Engineers (ASME) pressure vessels, control systemtheory, specific steps in one or more fermentation processes, and thelike have not been shown or described in detail to avoid unnecessarilyobscuring descriptions of the described embodiments. Unless the contextrequires otherwise, throughout the specification and claims whichfollow, the word “comprise” and variations thereof, such as, “comprises”and “comprising” are to be construed in an open, inclusive sense, thatis, as “including, but not limited to.” Further, headings providedherein are for convenience only and do not interpret the scope ormeaning of the claimed invention.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments. Also, as used in thisspecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the content clearly dictatesotherwise. It should also be noted that the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

Fermentors are generally defined as any vessel in which a fermentationprocess is carried out. Given the vast number of fermentation processesand the wide variety of fermentable substrates, fermentors can rangefrom simple continuous stirred tank reactors found in the alcoholicbeverage industry to highly complex, specialized vessels having gasdistribution and internal structures tailored to a particular substrateand/or a particular biological species. Fermentors useful in convertingcarbon-containing gases such as methane and syngas (a mixture of CO andH₂) to longer chain gaseous and liquid hydrocarbons generally disperse agas substrate containing the C₁ carbon compound within a liquid mediacontaining one or more nutrients to provide a multi-phase mixture. Thismulti-phase mixture is fed to one or more microbiological colonies thatconvert a portion of the C₁ carbon compound(s) in the gas substrate tomore preferred, longer chain, C₂ or higher compounds. The substratecomposition, nutrients, and microbiological organisms comprising thecolony (i.e., the biomass within the fermentor) can be variouslyadjusted or tailored to provide a desired final matrix of C₂ or highercompounds which may be present as a liquid, gas, or intracellularmaterial.

Fermentors useful in utilizing carbon-containing gases such as methaneand syngas (a mixture of CO and H₂) as a substrate for culturing singlecell microorganisms such as fungi, yeasts and bacteria which containhigh proportions of proteins generally disperse a gas substratecontaining a C₁ carbon compound within a liquid media containing one ormore nutrients to provide a multi-phase mixture. This multi-phasemixture is contacted with one or more microbiological colonies thatconvert a portion of the C₁ carbon compound(s) in the gas substrate toproteins. The substrate composition, nutrients, and microbiologicalorganisms comprising the colony (i.e., the biomass within the fermentor)can be variously adjusted or tailored to provide a desired final matrixof protein-containing biomass.

From a mass transfer perspective, gas substrate fermentors present aunique challenge in that the substrate is trapped within a gas bubbleand in order for microbiological uptake of the substrate to occur, thegas substrate must first pass from the gas bubble to the microbiologicalorganisms either directly or indirectly via dissolution in the liquidmedia. Such fermentation processes are thus frequently limited by theability of the system to facilitate and/or sustain a desirably highlevel of mass transfer of the substrate from the gas bubbles to themicrobiological organisms within the fermentor. At the least, the rateof mass transfer from the gas bubble to either the surrounding liquidmedia or to a microbiological organisms is a function of the gaspressure within the gas bubble, the volume to surface area ratio of thegas bubble, and the contact time of the gas bubble with the surroundingliquid or microbiological organisms. Increasing the pressure within thegas bubble or increasing the contact time of the gas bubble with thesurrounding liquid or microbiological organisms results in a highereffective mass transfer rate between the substrate and themicrobiological organisms. Decreasing the volume to surface area ratioof the gas bubble (i.e., reducing the diameter of the gas bubbles)results in a higher effective mass transfer rate between the gas bubbleand the surrounding liquid. Preferred fermentors from a mass transferstandpoint would therefore generate a large number of relatively smalldiameter gas bubbles at a relatively high pressure that are held inclose or intimate contact with the surrounding liquid or microbiologicalorganisms for an extended period of time.

Disclosed herein are a number of fermentation systems, methods, andapparatuses that are capable of providing relatively small diameter,relatively high pressure gas bubbles. Disclosed herein are a number offermentation systems, methods, and apparatuses capable of providing anextended contact time with the surrounding liquid and/or biologicalorganism(s). Such fermentation systems, methods, and apparatuses canadvantageously provide a highly efficient gas substrate fermentationsystem that may be particularly useful in converting C₁ compounds tomore preferred gaseous, liquid, and intra-cellular C₂ and highercompounds or stimulating the growth of microorganisms containing highproportions of protein.

As used herein, the terms “C₁ substrate” or “C₁ compound” refer to anycarbon-containing molecule or composition that lacks a carbon-carbonbond. Sample C₁ molecules or compositions include methane, methanol,formaldehyde, formic acid or a salt thereof, carbon monoxide, carbondioxide, syngas, methylamines (e.g., monomethylamine, dimethylamine,trimethylamine), methylthiols, or methylhalogens.

As used herein, the term “microorganism” refers to any microorganismhaving the ability to use a gaseous substrate as a source of energy oras its sole source of energy and biomass, and may or may not use othercarbon substrates (such as sugars and complex carbohydrates) for energyand biomass. Examples of microorganisms as used herein include theheterotrophic bacteria Ralstonia sp. (formerly Alcaligenes acidovorans)DB3 (strain NCIMB 13287), Brevibacillus agri (formerly Bacillus firmus)DB5 (strain NCIMB 13289) and Aneurinibacillus sp. (formerly Bacillusbrevis) DB4 (strain NCIMB 13288) which each have optimum growth at atemperature of about 45° C. Ralstonia sp. DB3 is a gram-negative,aerobic, motile rod belonging to the family Pseudomonadaceae which canuse ethanol, acetate, propionate and butyrate for growth.Aneurinibacillus sp. DB4 is a gram-negative, endospore-forming, aerobicrod belonging to the genus Bacillus which can utilize acetate,D-fructose, D-mannose, ribose and D-tagatose. Brevibacillus agri DB5 isa gram-negative, endospore-forming, motile, aerobic rod of the genusBacillus which can utilize acetate, N-acetyl-glucosamine, citrate,gluconate, D-glucose, glycerol and mannitol. Suitable yeasts for use inthe processes of the invention may be selected from the group consistingof Saccharomyces and Candida.

If desired, the processes described herein may be performed usingbacteria (or yeasts) genetically modified so as to generate a desiredchemical compound which can then be extracted from the intercellularfluid or the biomass harvested from the reactor. The scientific andpatent literature contains numerous examples of such geneticallymodified microorganisms including, inter alia, methanotrophic bacteria.

In at least some instances in accordance with embodiments describedherein, the microbiological organisms used to ferment gaseouscarbon-containing feedstocks employ a culture primarily comprising a C₁metabolizing non-photosynthetic microorganism. Such fermentation systemsmay use one or more species of C₁ metabolizing microorganisms that areprokaryotes or bacteria, such as Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium,Methanomonas, Methylophilus, Methylobacillus, Methylobacterium,Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia,Arthrobacter, Rhodopseudomonas, or Pseudomonas. In some instances, theC₁ metabolizing bacteria may include a methanotroph or a methylotroph.Preferred methanotrophs include Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium,Methanomonas, or a combination thereof. Exemplary methanotrophs includeMethylomonas sp. 16a (ATCC PTA 2402), Methylosinus trichosporium (NRRLB-ll,196), Methylosinus sporium (NRRL B-ll, 197), Methylocystis parvus(NRRL B-ll, 198), Methylomonas methanica (NRRL B-5 11,199), Methylomonasalbus (NRRL B-ll,200), Methylobacter capsulatus (NRRL B-11,201),Methylobacterium organophilum (ATCC 27,886), Methylomonas sp. AJ-3670(FERM P-2400), Methylomicrobium alcaliphilum Methylocella silvestris,Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylosinustrichosporium OB3b, Methylococcus capsulatus Bath, Methylomonas sp. 16a,Methylomicrobium alcaliphilum 20Z, or a high growth variants thereof.Preferred methylotrophs include Methylobacterium extorquens,Methylobacterium radiotolerans, Methylobacterium populi,Methylobacterium chloromethanicum, Methylobacterium nodulans, or acombination thereof.

Microorganisms capable of metabolizing C₁ compounds found in syngasinclude, but are not limited to Clostridium, Moorella, Pyrococcus,Eubacterium, Desulfobacterium, Carboxydothermus, Acetogenium,Acetobacterium, Acetoanaerobium, Butyribacterium, Peptostreptococcus, orcombinations thereof may also be used. Exemplary methylotrophs includeClostridium autoethanogenum, Clostridium ljungdahli, Clostridiumragsdalei, Clostridium carboxydivorans, Butyribacteriummethylotrophicum, Clostridium woodii, Clostridium neopropanologen, or acombination thereof. In some instances, C₁ metabolizing microorganismsare eukaryotes such as yeast, including Candida, Yarrowia, Hansenula,Pichia, Torulopsis, or Rhodotorula.

In other instances, the C₁ metabolizing non-photosynthetic microorganismis an obligate C₁ metabolizing non-photosynthetic microorganism, such asan obligate methanotroph or methylotroph. In some instances, the C₁metabolizing non-photosynthetic microorganism is a recombinantmicroorganism comprising a heterologous polynucleotide encoding a fattyacid producing enzyme, a formaldehyde assimilation enzyme, or acombination thereof.

As used herein, the terms “C₁ metabolizing microorganism” or “C₁metabolizing non-photosynthetic microorganism” refer to anymicroorganism having the ability to use a single carbon (C₁) substrateas a source of energy or as its sole source of energy and biomass, andmay or may not use other carbon substrates (such as sugars and complexcarbohydrates) for energy and biomass. For example, a C₁ metabolizingmicroorganism may oxidize a C₁ substrate, such as methane or methanol.C₁ metabolizing microorganisms include bacteria (such as Methanotrophsand Methylotrophs) and yeast. In at least some instances, a C₁metabolizing microorganism does not include a photosyntheticmicroorganism, such as algae. In certain embodiments, the C₁metabolizing microorganism will be an “obligate C₁ metabolizingmicroorganism,” meaning its sole source of energy comprises C₁substrates and nothing else.

As used herein, the term “methylotrophic bacteria” refers to anybacteria capable of oxidizing organic compounds that do not containcarbon-carbon bonds. In certain embodiments, a methylotrophic bacteriummay be a methanotroph. For example, “methanotrophic bacteria” refers toany methylotrophic bacteria that has the ability to oxidize methane asits primary source of carbon and energy. Exemplary methanotrophicbacteria include Methylomonas, Methylobacter, Methylococcus,Methylosinus, Methylocystis, Methylomicrobium, or Methanomonas. Incertain other embodiments, the methylotrophic bacterium is an “obligatemethylotrophic bacterium,” which refers to bacteria that are limited tothe use of C₁ substrates for the generation of energy.

In one specific embodiment of the invention, the process is performedusing methanotrophic bacteria of the type described in WO 02/18617 toproduce carotenoids, e.g., antheraxanthin, adonixanthin, astaxanthin,canthaxanthin, zeaxanthin and the other carotenoids mentioned on pages39 and 40 of WO 02/18617. To this end, the methanotrophic bacteriumMethylomonas 16a (ATCC PTA 2402) may particularly suitably be used.Carotenoids produced in this way may be separated out from the liquidculture medium as described in WO 02/18617, WO 02/20728 and WO 02/20733.

As used herein, the term “syngas” refers to a mixture including at leastcarbon monoxide (CO) and hydrogen (H₂). In at least some instances,syngas may also include CO₂, methane, and other gases in smallerquantities relative to CO and H₂. Syngas may be prepared using anyavailable process, including but not limited to, a water gas shift orcoal gasification process.

As used herein, the term “growth” is defined as any increase in cellmass. This may occur through cell division (replication) and theformation of new cells during “balanced growth,” or during “unbalancedgrowth” when cellular mass increases due to the accumulation of one ormore intracellular or intercellular polymers, such as certain lipids. Inthe latter case, growth may be manifest as an increase in cell size dueto the accumulation of a biopolymer within the cell. During “balancedcell growth,” all of the feedstocks (electron donors and electronacceptors) and all of the nutrients are present in the ratios requiredto make all of the macromolecular components of a cell. That is, nofeedstock or nutrient limits the synthesis of proteins, complexcarbohydrate polymers, fats, or nucleic acids. In contrast, during“unbalanced cell growth,” a feedstock or nutrient needed to make one ormore of a cell's macromolecules is not present in an amount or ratiorequired for balanced growth. Accordingly, this feedstock or nutrientbecomes limiting and is referred to as a “limiting nutrient.”

Some cells may still achieve net growth under unbalanced conditions, butthe growth is unbalanced and polymers that can be synthesized in theabsence of the limiting feedstock or nutrient will accumulate. Thesepolymers include lipids or intracellular storage products, such as thepolyhydroxyalkanoates (PHAs), including polyhydroxybutyrate (PHB),polyhydroxyvalerate (PHV), and polyhydroxyhexanoate (PHHx)-glycogen, orsecreted materials, such as extracellular polysaccharide. Such oilcompositions are useful in the production of bioplastics.

Sample balanced and unbalanced growth conditions may differ in thenitrogen content in the media. For example, nitrogen constitutes about12% of dry cell weight, which means that 12 mg/L nitrogen must besupplied (along with a feedstock and other nutrients in the requiredstoichiometric ratios) to grow 100 mg/L dry cell weight. If otherfeedstock and nutrients are available in the quantities needed toproduce 100 mg/L of dry cell weight, but less than 12 mg/L nitrogen isprovided, then unbalanced cell growth may occur, with accumulation ofpolymers that do not contain nitrogen. If nitrogen is subsequentlyprovided, the stored polymer may serve as feedstock for the cell,allowing balanced growth, with replication and production of new cells.

As used herein, the term “growth cycle” as applied to a cell ormicroorganism refers to the metabolic cycle through which a cell ormicroorganism moves in culture conditions. For example, the cycle mayinclude various stages, such as a lag phase, an exponential phase, theend of exponential phase, and a stationary phase.

As used herein, the term “exponential growth,” “exponential phasegrowth,” “log phase” or “log phase growth” refer to the rate at whichmicroorganisms are growing and dividing. For example, during log phase,microorganisms are growing at their maximal rate given their geneticpotential, the nature of the medium, and the conditions under which theyare grown. Microorganism rate of growth is constant during exponentialphase and the microorganism divides and doubles in number at regularintervals. Cells that are “actively growing” are those that are growingin log phase. In contrast, “stationary phase” refers to the point in thegrowth cycle during which cell growth of a culture slows or even ceases.

As used herein, the term “high growth variant” refers to an organism,microorganism, bacterium, yeast, or cell capable of growth with a C₁substrate, such as methane or methanol, as the sole carbon and energysource and which possesses an exponential phase growth rate that isfaster than the parent, reference or wild-type organism, microorganism,bacterium, yeast, or cell—that is, the high growth variant has a fasterdoubling time and consequently a high rate of growth and yield of cellmass per gram of C₁ substrate metabolized as compared to a parent cell(see, e.g., U.S. Pat. No. 6,689,601).

As used herein, the term “biofuel” refers to a fuel at least partiallyderived from “biomass.”

As used herein, the term “biomass” or “biological material” refers toorganic material having a biological origin, which may include one ormore of whole cells, lysed cells, extracellular material, or the like.For example, the material harvested from a cultured microorganism (e.g.,bacterial or yeast culture) is considered the biomass, which can includecells, cell membranes, cell cytoplasm, inclusion bodies, productssecreted or excreted into the culture medium, or any combinationthereof. In certain embodiments, biomass comprises the C₁ metabolizingmicroorganisms of this disclosure together with the media of the culturein which the C₁ metabolizing microorganisms of this disclosure weregrown. In other embodiments, biomass comprises C₁ metabolizingmicroorganisms (whole or lysed or both) of this disclosure recoveredfrom a culture grown on a C₁ (e.g., natural gas, methane). In stillother embodiments, biomass comprises the spent media supernatant orgases excreted or secreted from a culture of C₁ metabolizingmicroorganism culture on a C₁ substrate. Such a culture may beconsidered a renewable resource.

As used herein, the term “biorefinery” refers to a facility thatintegrates biomass conversion processes and equipment to produce fuelsfrom biomass.

As used herein, “oil composition” refers to the lipid content of abiomass (e.g., bacterial culture), including fatty acids, fatty acidesters, triglycerides, phospholipids, poly hydroxyalkanoates, isoprenes,terpenes, or the like. In oil composition of a biomass may be extractedfrom the rest of the biomass materials, such as by hexane or chloroformextraction. In addition, an “oil composition” may be found in any one ormore areas of a culture, including the cell membrane, cell cytoplasm,inclusion bodies, she treated or excreted into the culture medium, orany combination thereof. An oil composition is neither natural gas norcrude petroleum.

As used herein, the term “refinery” refers to an oil refinery, oraspects thereof, at which oil compositions (e.g., biomass, biofuel, orfossil fuels such as crude oil, coal or natural gas) may be processed.Sample processes carried out at such refineries include cracking,transesterification, reforming, distilling, hydroprocessing,isomerization, or any combination thereof.

As used herein, the terms “recombinant” or “non-natural” refer to anorganism microorganism, cell, nucleic acid molecule, or vector that hasat least one genetic alteration or has been modified by the introductionof a heterologous nucleic acid molecule, or refers to a cell that hasbeen altered such that the expression of an endogenous nucleic acidmolecule or gene can be controlled. Recombinant also refers to a cellthat is derived from a cell having one or more such modifications. Forexample, recombinant cells may express genes or other nucleic acidmolecules that are not found in identical form within the native cell(i.e., unmodified or wild type cell), or may provide an alteredexpression pattern of endogenous genes, such genes that may otherwise beover-expressed, under-expressed, minimally expressed, or not expressedat all. In another example, genetic modifications to nucleic acidmolecules encoding enzymes or functional fragments thereof can providebiochemical reaction(s) or metabolic pathway capabilities to arecombinant microorganism or cell that is new or altered from itsnaturally occurring state.

As used herein, the term “heterologous” nucleic acid molecule, constructor sequence refers to a nucleic acid molecule or portion of a nucleicacid molecule sequence that is not native to a cell in which it isexpressed or is a nucleic acid molecule with an altered expression ascompared to the native expression levels in similar conditions. Forexample, a heterologous control sequence (e.g., promoter, enhancer) maybe used to regulate expression of a gene or a nucleic acid molecule in away that is different than the gene or a nucleic acid molecule isnormally expressed in nature or culture. Generally, heterologous nucleicacid molecules are not endogenous to the cell or part of the genome inwhich they are present, and have been added to the cell by conjugation,transformation, transfection, electroporation, or the like.

As used herein, the term “vertical” refers to a direction that isaligned with the gravity vector at the location in question.

As used herein, the term “horizontal” refers to a direction that isperpendicular to the gravity vector at the location in question.

As used herein, the term “non-vertical” refers to a direction that ishorizontal (i.e., perpendicular to vertical) or 20° or more fromvertical, e.g., more than 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°,65°, 70°, 75°, 80° or 85° from vertical.

The systems for fermentation of the instant disclosure may includeseparate units (e.g., processing units or systems that are disposed inclose proximity or adjacent to each other, or not), integrated units, orthe system itself may be interconnected and integrated. The systems ofthis disclosure may use at least one gas phase feedstock, including oneor more C₁ compounds, oxygen, and/or hydrogen. In certain embodiments,the fermentation system uses a C₁ metabolizing microorganism (e.g., amethanotroph such as Methylosinus trichosporium OB3b, Methylococcuscapsulatus Bath, Methylomonas sp. 16a, Methylomicrobium alcaliphilum20Z, or high growth variants or combinations thereof) as the primarymicroorganism in the fermentation culture.

A variety of culture methodologies may be used for the microorganism,bacteria and yeast described herein. For example, C₁ metabolizingmicroorganisms, such as methanotroph or methylotroph bacteria, may begrown by batch culture and continuous culture methodologies. Generallycells in log phase are often responsible for the bulk production of aproduct or intermediate of interest in some systems, whereas stationaryor post-exponential phase production can be obtained in other systems.

A classical batch culturing method is a closed system in which the mediacomposition is set when the culture is started and is not altered duringthe culture process. That is, media is inoculated at the beginning ofthe culturing process with one or more microorganisms of choice and thenis allowed to grow without adding additional microorganisms to thesystem. As used herein, a “batch” culture is in reference to notchanging the amount of a particular carbon source initially added,whereas control of factors such as pH and oxygen and/or hydrogenconcentration can be monitored and altered during the culture. In batchsystems, metabolite and biomass compositions of the system changeconstantly up to the time the culture is terminated. Within batchcultures, cells (e.g., bacteria such as methylotrophs) will generallymove from a static lag phase to a high growth logarithmic phase to astationary phase where growth rate is reduced or stopped (and willeventually lead to cell death if conditions do not change).

A fed-batch system is a variation on the standard batch system in whicha carbon substrate of interest is added in increments as the cultureprogresses. Fed-batch systems are useful when cell metabolism is likelyto be inhibited by catabolite repression and when it is desirable tohave limited amounts of substrate in the media. Since it is difficult tomeasure actual substrate concentration in fed-batch systems, an estimateis made based on changes of measurable factors such as pH, dissolvedoxygen, and the partial pressure of waste gases. Batch and fed-batchculturing methods are common and known in the art (see, e.g., Thomas D.Brock, Biotechnology: A Textbook of Industrial Microbiology, 2nd Ed.(1989) Sinauer Associates, Inc., Sunderland, Mass.; Deshpande, 1992,Appl. Biochem. Biotechnol. 36:227).

Continuous cultures are “open” systems in the sense that defined culturemedia is continuously added to a bioreactor while an equal amount ofused (“conditioned”) media is removed simultaneously for processing.Continuous cultures generally maintain the cells at a constant high,liquid phase density where cells are primarily in logarithmic growthphase. Alternatively, continuous culture may be practiced withimmobilized cells (e.g., biofilm) where carbon and nutrients arecontinuously added and valuable products, by-products, and wasteproducts are continuously removed from the cell mass. Cellimmobilization may be achieved with a wide range of solid supportscomposed of natural materials, synthetic materials, or a combinationthereof.

Continuous or semi-continuous culture allows for the modulation of oneor more factors that affect cell growth or end product concentration.For example, one method may maintain a limited nutrient at a fixed rate(e.g., carbon source, nitrogen) and allow all other parameters to changeover time. In other embodiments, several factors affecting growth may becontinuously altered while cell concentration, as measured by mediaturbidity, is kept constant. The goal of a continuous culture system isto maintain steady state growth conditions while balancing cell loss dueto media being drawn off against the cell growth rate. Methods ofmodulating nutrients and growth factors for continuous culture processesand techniques for maximizing the rate of product formation are wellknown in the art (see Brock, 1992).

In certain embodiments, culture media includes a carbon substrate as asource of energy for a C₁ metabolizing microorganism. Suitablesubstrates include C₁ substrates, such as methane, methanol,formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide,methylated amines (methylamine, dimethylamine, trimethylamine, etc.),methylated thiols, or methyl halogens (bromomethane, chloromethane,iodomethane, dichloromethane, etc.). In certain embodiments, culturemedia may comprise a single C₁ substrate as the sole carbon source for aC₁ metabolizing microorganism, or may comprise a mixture of two or moreC₁ substrates (mixed C₁ substrate composition) as multiple carbonsources for a C₁ metabolizing microorganism.

Additionally, some C₁ metabolizing organisms are known to utilize non-C₁substrates, such as sugar, glucosamine or a variety of amino acids formetabolic activity. For example, some Candida species can metabolizealanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489,1990). Methylobacterium extorquens AM1 is capable of growth on a limitednumber of C₂, C₃, and C₄ substrates (Van Dien et al., Microbiol.149:601-609, 2003). Alternatively, a C₁ metabolizing microorganism maybe a recombinant variant having the ability to utilize alternativecarbon substrates. Hence, it is contemplated that a carbon source inculture media may comprise a mixture of carbon substrates, with singleand multi-carbon compounds, depending on the C₁ metabolizingmicroorganism selected.

In certain embodiments, the instant disclosure provides a method formaking fuel, comprising converting biomass from a culture primarilycomprising a C₁ metabolizing non-photosynthetic microorganism into anoil composition and refining the oil composition into a fuel. In certainembodiments, the C₁ metabolizing non-photosynthetic microorganism is anobligate C₁ metabolizing non-photosynthetic microorganism, such as anobligate methanotroph or methylotroph. In further embodiments, the C₁metabolizing non-photosynthetic microorganism is a recombinantmicroorganism comprising a heterologous polynucleotide encoding a fattyacid producing enzyme, a formaldehyde assimilation enzyme, or acombination thereof. In further embodiments, the oil composition isderived or extracted from cell membrane of the C₁ metabolizingnon-photosynthetic microorganism, such as a methylotroph ormethanotroph.

In certain embodiments, the instant disclosure provides a method formaking fuel by refining an oil composition in a refining unit to producefuel, wherein the oil composition is derived from a C₁ metabolizingnon-photosynthetic microorganism, such as a methylotroph ormethanotroph. In further embodiments, the method further comprises useof a processing unit for extracting the oil composition from the C₁metabolizing non-photosynthetic microorganism. In still furtherembodiments, the method comprises (a) culturing C₁ metabolizing bacteriain the presence of a feedstock comprising a C₁ substrate in a controlledculturing unit, wherein the cultured bacteria produces an oilcomposition; (b) extracting the oil composition from the culturedbacteria in a processing unit; and (c) refining the extracted oilcomposition in a refining unit to produce fuel. In certain embodiments,the feedstock C₁ substrate is methane, methanol, formaldehyde, formicacid, carbon monoxide, carbon dioxide, a methylamine, a methylthiol, ora methylhalogen.

In certain embodiments, the instant disclosure provides a method formaking natural products, such as ethanol, acetate, butanol, single-cellprotein, sugars, or other metabolites or cellular products wherein thenatural product is derived from a C₁ metabolizing non-photosyntheticmicroorganism, such as a methylotroph or methanotroph.

In further embodiments, the method further comprises use of a processingunit for extracting the natural product from the C₁ metabolizingnon-photosynthetic microorganism.

In still further embodiments, the method comprises (a) culturing C₁metabolizing bacteria in the presence of a feedstock comprising a C₁substrate in a controlled culturing unit, wherein the cultured bacteriaproduce a natural product; (b) extracting the natural product from thecultured bacteria in a processing unit; and (c) refining the naturalproduct to produce a commercial product. In certain embodiments, thefeedstock C₁ substrate is methane, methanol, formaldehyde, formic acid,carbon monoxide, carbon dioxide, a methylamine, a methylthiol, or amethylhalogen.

In certain embodiments, the instant disclosure provides a method formaking natural or non-natural products, such as ethanol, acetate,butanol, isoprene, propylene, farnesene, enzymes, or other metabolitesor cellular products wherein the product is derived from a geneticallyengineered C₁ metabolizing non-photosynthetic microorganism, such as amethylotroph or methanotroph which has been transformed with aheterologous nucleotide sequence. In further embodiments, the methodfurther comprises use of a processing unit for extracting the productfrom the genetically engineered C₁ metabolizing non-photosyntheticmicroorganism. In still further embodiments, the method comprises (a)culturing genetically engineered C₁ metabolizing bacteria in thepresence of a feedstock comprising a C₁ substrate in a controlledculturing unit, wherein the cultured bacteria produce a natural product;(b) extracting the natural product from the cultured bacteria in aprocessing unit; and (c) refining the natural product to produce acommercial product. In certain embodiments, the feedstock C₁ substrateis methane, methanol, formaldehyde, formic acid, carbon monoxide, carbondioxide, a methylamine, a methylthiol, or a methylhalogen.

In certain embodiments, the instant disclosure provides a method formaking natural or non-natural products, such as ethanol, acetate,butanol, isoprene, propylene, farnesene, enzymes, or other metabolitesor cellular products wherein the product is derived from a non-C₁metabolizing microorganism, such as Escherichia coli, Saccaromycescerevisiae, or other common production microorganism. In certainembodiments, the feedstock substrate is glucose, sucrose, glycerol,cellulose or other multicarbon feedstocks.

A loop reactor illustrated in FIG. 1 of U.S. Pat. No. 7,579,163 isdescribed as including a substantially vertical downflow zone 3 and asubstantially vertical upflow zone 5 separated by a substantiallyhorizontal zone 4 which begins at the bottom of the substantiallyvertical downflow zone 3 and ends at the beginning of substantiallyvertical upflow zone 5. The presence of the substantially verticaldownflow zone 3 and the substantially vertical upflow zone 5 results ina vertical distance between the gas liquid surface 22 at the end ofoutflow zone 6 and the centerline of the loop reactor in the horizontalzone 4. The '163 patent describes that this vertical distance is atleast 10 meters or about 32.8 feet. The distance liquid medium flowsupward through vertical upflow section 5 to a location where it entershorizontal effluent gas/liquid reaction medium separation section 6depends on the rise in the substantially horizontal section 4 of loopand the rise in the substantially horizontal effluent gas/liquidreaction medium separation section 6. The presence of a substantiallyvertical downflow zone and a substantially vertical upflow zone ofsufficient length to accommodate a vertical distance between the gasliquid surface at the end of an outflow zone 6 and the centerline of theloop reactor in the horizontal zone 4 on the order of 10 meterscontributes significantly to the overall cost of designing andmanufacturing a loop reactor with these zones. For example, the costsassociated with designing and manufacturing structures required tophysically support downflow and upflow vertical zones tall enough toaccommodate vertical distances between the gas liquid surface 22 at theend of outflow zone 6 and the centerline of the loop reactor in thehorizontal zone 4 on the order of 10 meters contributes significantly tothe overall cost of designing, building and maintaining a loop reactorincluding such zones. Loop reactors with upflow and downflow verticalzones on the order of 10 meters tall require buildings in which suchreactors are housed to have sufficient vertical clearance to accommodatesuch tall vertical upflow and downflow zones. A loop reactor of the typedescribed in the '163 patent which includes a vertical distance betweenthe gas liquid surface 22 at the end of outflow zone 6 and thecenterline of the loop reactor in the horizontal zone 4 of at least 10meters exhibits a static head or hydrostatic pressure in thesubstantially vertical downflow zone which is represented by the formulaP=ρg h, wherein P is the hydrostatic pressure in pascals, ρ is the fluiddensity in kg/m³, g is the gravitational acceleration in m/s² and h isthe length in meters of the vertical distance between the gas liquidsurface 22 at the end of outflow zone 6 and the centerline of the loopreactor in the horizontal zone 4. For a loop reactor of the typedescribed in the '163 patent which includes a vertical distance betweenthe gas liquid surface 22 at the end of outflow zone 6 and thecenterline of the loop reactor in the horizontal zone 4 of at least 10meters, the hydrostatic pressure P at the bottom of the substantiallyvertical downflow zone 6 can be characterized as being at least 10 ρg.The pressure on the inlet side of propeller 10 is the sum of thishydrostatic pressure P and the pressure in the effluent gas removalzone/top unit 2.

FIG. 2 shows an exemplary system 200 for stimulating production ofbiomass that includes a loop reactor 101 along with a separationsubsystem 250, an optional thermal subsystem 270 and optional controlsubsystem 290. Although shown as an integrated system 200, the optionalsubsystems may be installed or otherwise combined with the loop reactor101 either individually or in any combination. One or more liquids andone or more gas substrates are introduced to the loop reactor 101 toform a multi-phase mixture with a liquid culture media that travelsthrough the loop reactor 101. After passage through the loop reactor101, the multi-phase mixture may contain one or more compounds producedby the biological organisms within the loop reactor 101, unconsumednutrients and other compounds in the liquid within the multi-phasemixture, unconsumed gases in the gas bubbles within the multi-phasemixture, and microbiological organisms in the form of biosolids. Excessmicrobiological organisms may be removed from the loop reactor 101 asbiomass either intermittently or continuously. Biomass accumulationswithin the loop reactor 101 may be removed to maintain the overallbiomass within the loop reactor 101 within a defined range or above orbelow a defined threshold. In at least some instances, biomass removedfrom the loop reactor 101 may include one or more useful compounds. Forexample, the biological organisms within the excess biomass may containan amount of one or more intracellular lipids or similar compoundsuseful in the production of a biofuel such as biodiesel orprotein-containing products.

The one or more liquids may include any liquid suitable for sustainingor delivering one or more nutrients to the microbiological organismswithin the loop reactor 101. Such liquids may include, but are notlimited to, solutions containing water, one or more alcohols, minerals,one or more nitrogen-containing compounds, one or morephosphorus-containing compounds, and the like. In at least someinstances, one or more fluid movers are used to deliver the one or moreliquids to the loop reactor 101 in a controlled manner and pressure. Theone or more fluid movers can include any type of pump or similar devicecapable of transferring a liquid between two points. Example fluidmovers include, but are not limited to, centrifugal pumps, positivedisplacement pumps, progressing cavity pumps, double diaphragm pumps,and the like. Other illustrative fluid movers include, but are notlimited to eductors, ejectors, and similar devices. The transfer ofliquid to the loop reactor 101 can be flow controlled, pressurecontrolled, or controlled using combinations of pressure, temperature,flow, level, flowrate, superficial velocity, or compositional analysisprocess variable data gathered from one or more points within the loopreactor 101 or from one or more points within the system 200. In atleast some instances, the transfer of liquid by the fluid mover can becontrolled based on the measured concentration of one or more componentsor compounds (e.g., one or more carbon-containing or nitrogen-containingnutrients) within the loop reactor 101; for example, the flow of liquidtransferred by the fluid mover may be increased in response to ameasured decrease in nutrient concentration within the loop reactor 101.

The one or more gas substrates can include any gas, gases, orcombination of gases suitable for sustaining or delivering one or morenutrients to the biological organisms within the loop reactor 101. Suchgases can include, but are not limited to, one or more gases containingcarbon compounds. Such gases can include, but are not limited to, one ormore gases containing C₁ carbon compounds such as methane or carbonmonoxide. The one or more gas substrates may also include one or moregases used in the metabolic processes of the biological organisms withinthe loop reactor 101. Such gases can include, but are not limited to,oxygen, oxygen-containing compounds and hydrogen. The one or more gassubstrates may be transferred to the loop reactor 101 as a pure gas oras a gas mixture (e.g., syngas, a mixture of carbon monoxide andhydrogen). The one or more gas substrates may be transferred to the loopreactor 101 individually (e.g., methane and an oxygen-containing gassuch as air may be transferred individually to minimize the likelihoodof formation of an explosive gas mixture external to the loop reactor101).

The one or more gas substrates may optionally be transferred to the loopreactor 101 using a gas mover. Example gas movers include, but are notlimited to, rotary lobe compressors, centrifugal compressors, screwcompressors, and the like. The delivery pressure of the one or more gassubstrates depends upon a variety of factors including the operatingpressure of the loop reactor 101 and the pressure drop associated withthe gas distributor used to distribute the one or more gas substrateswithin the loop reactor 101. Similarly, the delivery flowrate of the oneor more gas substrates may be manually or automatically controlled tomaintain the concentration or level of dissolved gas within the loopreactor 101 within a defined range (e.g., dissolved oxygen above atleast 4 ppm) based at least in part on the needs of the biologicalorganisms present in the loop reactor 101. In at least some instances,the one or more gas substrates can be delivered to the loop reactor 101at a pressure of from about 1.5 psig to about 600 psig, about 5 psig toabout 600 psig; from about 25 psig to about 400 psig; or from about 50psig to about 300 psig.

Any number of gases may be introduced through a common gas distributionheader or any number of individual gas distribution headers. Such gasdistribution headers may introduce all of the gas substrate at a singlepoint within the loop reactor 101 or may introduce portions of the gassubstrate at various locations throughout the loop reactor 101. In atleast some instances, the gas substrate can include, but is not limitedto, methane, carbon monoxide, hydrogen, or oxygen. In at least someinstances, the feed rate of the gas substrate can be referenced to thefeed rate of the liquid media. For example, methane may be introduced asa gas substrate at a rate of from about 0.1 grams of methane/liter ofliquid media (g/l) to about 100 g/l; from about 0.5 g/l to about 50 g/l;or from about 1 g/l to about 25 g/l. Carbon monoxide (“CO”) may beintroduced as a gas substrate 204 at a rate of from about 0.1 grams ofCO/liter of liquid media (g/l) to about 100 g/l; from about 0.5 g/l toabout 50 g/l; or from about 1 g/l to about 25 g/l. Oxygen may beintroduced as a gas substrate 204 at a rate of from about 1 grams ofoxygen/liter of liquid media (g/l) to about 100 g/l; from about 2 g/l toabout 50 g/l; or from about 5 g/l to about 25 g/l. Hydrogen may beintroduced as a gas substrate 204 at a rate of from about 0.01 grams ofhydrogen/liter of liquid media (g/l) to about 50 g/l; from about 0.1 g/lto about 25 g/l; or from about 1 g/l to about 10 g/l.

Within the loop reactor 101 the microbiological organisms willmetabolize at least a portion of the carbon-containing compounds presentin the multi-phase mixture. At least a portion of this process mayinclude the production of additional microbiological organisms thatincrease the overall quantity of biomass present in the loop reactor101. Left uncontrolled, the biomass within the loop reactor 101 mayaccumulate to a point such that one or more operational aspects of theloop reactor 101 (e.g., flowrate, pressure drop, production of desiredproducts, etc.) is compromised or adversely affected by the presence ofthe excess biomass. In such instances, the ability to remove at least aportion of the biomass present in the loop reactor 101 is desirable. Inat least some instances, biomass preferentially accumulates at alocation within a gas/liquid separation vessel (102 in FIGS. 3 and 4)facilitating biosolids removal from the loop reactor 101 via the atleast one biomass removal port (128 in FIGS. 3 and 4). The removedbiomass can be delivered to separation subsystem 250 where the biomasscan be further processed and desirable products recovered from thebiomass.

In at least some instances, all or a portion of the biomass productionprocess may be at least partially automatically controlled using acontrol subsystem 290. The control subsystem 290 may collectprocess-related information provided by one or more process elements inthe form of signals containing analog or digital data representing oneor more process variables. For instance, the control subsystem cancollect process-related signals using one or more process elementsincluding, but not limited to, mass flow sensors, volumetric flowsensors, temperature sensors, pressure sensors, level sensors,analytical sensors (e.g., dissolved oxygen sensors, biological oxygendemand or “BOD” sensors, pH sensors, conductivity sensors, and the like)or any other device capable of providing a signal containing datarepresentative of one or more process-related conditions within the loopreactor 101.

The control subsystem 290 may execute one or more sets of instructionscontrolling, altering, or adjusting one or more aspects of thefermentation process based at least in part on the process variablesignals received from the process elements. Such instructions may resultin the generation of one or more control output signals by the controlsubsystem 290. The control output signals can be transmitted from thecontrol subsystem 290 to one or more final control elements such asblock valves, control valves, motors, variable speed drives, etc. Theinteraction between the final control elements and the fermentationprocess can, in turn, provide the control subsystem 290 with a highdegree of relatively accurate control of the biomass production process.

For example, responsive to the receipt of one or more signals containingdata indicative of the temperature of the multi-phase mixture in theloop reactor 101, the control subsystem 290 may initiate, alter, orcease the flow of thermal transfer media to a heat transfer unitoperation. Similarly, responsive to the receipt of one or more signalscontaining data indicative of the dissolved oxygen level of themulti-phase mixture in the loop reactor 101, the control subsystem 290may increase, decrease, or maintain the flow of the oxygen-containinggas substrate to the loop reactor 101. Although only two illustrativeexamples are provided herein, any flow, level, pressure, analyticalvalue, or the like that is appropriate to the fermentation process maybe similarly controlled by the control subsystem 290 using one or moreappropriate process sensors and one or more appropriate final controlelements.

FIGS. 3 and 4 show an exemplary system 100 for stimulating production ofbiomass. Exemplary system 100 includes a loop reactor 101 including agas/liquid separation unit operation 102 (e.g., a gas/liquid separationvessel or other equipment capable of separating liquids and gases from amulti-phase mixture of liquid culture media including microorganisms anda fluid flow unit operation 104 (e.g., pump or other device capable ofcausing a fluid to move), a loop section 106 and a first non-verticalpressure reduction zone 108. As used herein, the loop section 106 refersto that portion of loop reactor 101 extending from the outlet of fluidflow unit operation 104 to the gas/liquid separation unit operation 102.Loop section 106 may or may not include vertical portions. When loopsection 106 does not include vertical portions, it can be referred to asa non-vertical loop section 106. In additional embodiments of theexemplary system 100, loop reactor 101 includes a second pressurereduction zone 112 (illustrated in FIG. 3) downstream of the firstnon-vertical pressure reduction zone 108. In additional exemplaryembodiments, second pressure reduction zone 112 may be a secondnon-vertical pressure reduction zone or it may be a vertical pressurereduction zone. A vertical pressure reduction zone 147 is illustrated inFIG. 3. Exemplary system 100 in additional embodiments includes othersubsystems, including nutrient and/or mineral supply subsystem 114 andheat transfer unit operation(s) 116. Exemplary system 100 stimulatesproduction of biomass by introducing gaseous substrate(s) andnutrient(s) to a liquid culture medium to form a multi-phase mixture ofthe liquid culture medium, supplied gaseous substrate(s) andnutrient(s). This multi-phase mixture flows through loop reactor 101 bythe action of fluid flow unit operation 104. The liquid culture mediumincludes microorganisms capable of converting gaseous substrates todesirable products, some of which may be recovered from themicroorganisms or from the gas phase and/or liquid phase that form ingas/liquid separation unit operation 102. Gaseous substrate(s) andnutrient(s) can be delivered to loop reactor 101 from nutrient supplysubsystem 114, and loop reactor 101 is operated under conditions thatpromote mass transfer of gaseous substrate(s) and nutrient(s) into theliquid culture medium and into the microorganisms. Nutrients andminerals can be introduced at locations other than as indicated bynutrient/mineral supply subsystem 114. For example, minerals and/ornutrients may be supplied at heat transfer unit operation(s) 116.Gas/liquid separation vessel 102 receives the liquid culture medium,including any gases that remain in the liquid culture medium, and gaseswhich have separated from the liquid culture medium, and separates theminto a liquid phase and a gas phase. The liquid phase separated from thegas phase in gas/liquid separation vessel 102 is removed from gas/liquidseparation vessel 102 and received by fluid flow unit operation 104.

Exemplary system 100 illustrated in FIG. 4 includes a loop section 106that does not include any vertical sections. Exemplary system 100illustrated in FIG. 3 includes a loop section 106 that includes avertical section shorter than vertical sections included in loopsections of conventional loop reactors. For example, loop section 106 ofexemplary system 100 in FIG. 3 can include a vertical section that is nomore than 50%, no more than 40%, no more than 30%, no more than 20% orno more than 10% of the vertical distance between centerline of loopsection 106 at its outlet 135 (i.e., at the inlet to the gas/liquidseparation unit operation 102) and the centerline of loop section 106 atthe outlet 131 of fluid flow unit operation 104. Referring to FIGS. 3and 4, the portion of the loop reactor between the gas/liquid interface118 within gas/liquid separation unit operation 102 and the centerlineof loop section 106 at outlet 131 of fluid flow unit operation 104 is asubstantially vertical downflow zone. The vertical distance between thegas/liquid interface 118 in gas/liquid separation unit operation 102 andthe centerline of loop section 106 at outlet 131 of fluid flow unitoperation 104 is equal to the vertical distance between the loop sectioncenterline of loop section 106 at its outlet 135 and the centerline ofloop section 106 at outlet 131 of fluid flow unit operation 104 when thegas/liquid interface 118 in gas/liquid separation unit operation 102coincides with (i.e., is at the same elevation as) the loop sectioncenterline of loop section 106 at its outlet 135. In other embodiments,the gas/liquid interface 118 in gas/liquid separation unit operation 102is below the loop section centerline of loop section 106 at its outlet135 and does not coincide with the loop section centerline of loopsection 106 at its outlet 135. In these embodiments, the verticaldistance between the gas/liquid interface 118 in gas/liquid separationunit operation 102 and the centerline of loop section 106 at outlet 131of fluid flow unit operation 104 is less than the vertical distancebetween the loop section centerline of loop section 106 at its outlet135 and the centerline of loop section 106 at outlet 131 of fluid flowunit operation 104. Exemplary system 100 is characterized by a verticaldistance between the centerline of loop section 106 at its outlet 135(i.e., at the inlet to the gas/liquid separation unit operation 102) andthe centerline of loop section 106 at outlet 131 of fluid flow unitoperation 104 (and the centerline of fluid flow unit operation 104 whenthe centerline of fluid flow unit operation 104 is at the same elevationas the centerline of loop section 106 at outlet 131 of fluid flow unitoperation 104) that is less than ten meters, less than nine meters, lessthan eight meters, less than seven meters, less than six meters, lessthan five meters, less than four meters, less than three meters, lessthan two meters, or less than one meter. In accordance with the abovedescribed embodiment, such loop reactors exhibit a static head orhydrostatic pressure upstream of fluid flow unit operation 104 at theinlet of the fluid flow unit operation which is represented by theformula P=ρg h, wherein P is the hydrostatic pressure in pascals, ρ isthe fluid density kg/m³, g is the gravitational acceleration in m/s² andh is the length in meters of the vertical distance between thegas/liquid interface 118 in gas/liquid separation unit operation 102 andthe centerline of loop section 106 at outlet 131 of fluid flow unitoperation 104. For loop reactors in accordance with the aboveembodiments, which includes a vertical distance between the gas/liquidinterface 118 in gas/liquid separation unit operation 102 and thecenterline of loop section 106 at outlet 131 of fluid flow unitoperation 104 that is less than 10 meters, the hydrostatic pressure P atthe inlet of fluid flow unit operation 104 which is at substantially thesame elevation as the centerline of loop section 106 at outlet 131 offluid flow unit operation 104 can be characterized as being less than 10ρg. The hydrostatic pressure P at the inlet of the fluid flow unitoperation 104 where the vertical distance between the gas/liquidinterface 118 in gas/liquid separation unit operation 102 and thecenterline of loop section 106 at outlet 131 of fluid flow unitoperation 104 is less than nine meters, less than eight meters, lessthan seven meters, less than six meters, less than five meters, lessthan four meters, less than three meters, less than two meters, or lessthan one meter long can be characterized as being less than 9 ρg, 8 ρg,7 ρg, 6 ρg, 5 ρg, 4 ρg, 3 ρg, 2 ρg or ρg, respectively. The pressure onthe inlet side of fluid flow unit operation 104 is the sum of thishydrostatic pressure P and the pressure in the headspace of gas/liquidseparation unit operation 102.

In those embodiments of exemplary system 100 where the gas/liquidinterface 118 is below the loop section centerline of loop section 106at its outlet and does not coincide with the loop section centerline ofloop section 106 at its outlet 135 and the vertical distance between thegas/liquid interface 118 in gas/liquid separation unit operation 102 andthe centerline of loop section 106 at outlet 131 of fluid flow unitoperation 104 that is less than 10 meters, the hydrostatic pressure P atthe inlet to the fluid flow unit operation 104 can be characterized asbeing less than 10 ρg. The hydrostatic pressure P at the inlet to thefluid flow unit operation 104 where the vertical distance between thegas/liquid interface 118 in gas/liquid separation unit operation 102 andthe centerline of loop section 106 at outlet 131 of fluid flow unitoperation 104 is less than nine meters, less than eight meters, lessthan seven meters, less than six meters, less than five meters, lessthan four meters, less than three meters, less than two meters, or lessthan one meter long can be characterized as being less than 9 ρg, 8 ρg,7 ρg, 6 ρg, 5 ρg, 4 ρg, 3 ρg, 2 ρg or ρg, respectively.

As noted above, the pressure on the inlet side of fluid flow unitoperation 104 is the sum of hydrostatic pressure P and the pressure inthe headspace of gas/liquid separation unit operation 102. In exemplaryembodiments described herein, the pressure at the inlet of fluid flowunit operation 104 which is at substantially the same elevation as thecenterline of loop section 106 at outlet 131 is less than 9 ρg+pressurein the headspace of gas/liquid separation unit operation 102, 8ρg+pressure in the headspace of gas/liquid separation unit operation102, 7 ρg+pressure in the headspace of gas/liquid separation unitoperation 102, 6 ρg+pressure in the headspace of gas/liquid separationunit operation 102, 5 ρg+pressure in the headspace of gas/liquidseparation unit operation 102, 4 ρg+pressure in the headspace ofgas/liquid separation unit operation 102, 3 ρg+pressure in the headspaceof gas/liquid separation unit operation 102, 2 ρg+pressure in theheadspace of gas/liquid separation unit operation 102 or ρg+pressure inthe headspace of gas/liquid separation unit operation 102 for systems100 where the vertical distance between the gas/liquid interface 118 ingas/liquid separation unit operation 102 and the centerline of loopsection 106 at outlet 131 of fluid flow unit operation 104 is less thannine meters, less than eight meters, less than seven meters, less thansix meters, less than five meters, less than four meters, less thanthree meters, less than two meters, or less than one meter,respectively. Exemplary pressure at the inlet to fluid flow unitoperation 104 are less than 0.9 bar gauge, less than 0.8 bar gauge, lessthan 0.7 bar gauge, less than 0.6 bar gauge, less than 0.5 bar gauge,0.4 bar gauge, less than 0.3 bars gauge, less than 0.2 bars gauge orless than 0.1 bars gauge. For example, pressure at the inlet to fluidflow unit operation 104 ranges from 0.55 bar gauge to 1.0 bar gauge,from 0.55 bar gauge to 0.8 bar gauge or from 0.55 bar gauge to 0.7 bargauge.

Loop reactors 101 in accordance with embodiments described hereininclude ratios of the length of the loop section 106 to the verticaldistance between the gas/liquid interface 118 in gas/liquid separationunit operation 102 and the centerline of loop section 106 at outlet 131of fluid flow unit operation 104 that are between 20:1 to 60:1 orbetween 30:1 to 50:1. Loop reactors in accordance with embodimentsdescribed herein are not limited to loop reactors that include ratios ofthe length of the loop section 106 to the vertical distance between thegas/liquid interface 118 in gas/liquid separation unit operation 102 andthe centerline of loop section 106 at outlet 131 of fluid flow unitoperation 104 that are between 20:1 to 60:1 or between 30:1 to 50:1.Loop reactors in accordance with embodiments described herein caninclude ratios of the length of the loop section 106 to the verticaldistance between the gas/liquid interface 118 in gas/liquid separationunit operation 102 and the centerline of loop section 106 at outlet 131of fluid flow unit operation 104 that fall outside the ranges of between20:1 to 60:1 or between 30:1 to 50:1. For example, loop reactors inaccordance with embodiments described herein have ratios of the lengthof the loop section 106 to the vertical distance between the gas/liquidinterface 118 in gas/liquid separation unit operation 102 and thecenterline of loop section 106 at outlet 131 of fluid flow unitoperation 104 that are less than 20:1 or greater than 60:1. For exampleloop reactors 101 in accordance with embodiments described herein canhave ratios that are greater than 60:1, e.g., ratio up to 100:1 or more.

Elements of loop reactor 101 including but not limited to gas/liquidseparation unit operation 102 (e.g., a gas/liquid separation vessel orother equipment capable of separating liquids and gases from amulti-phase mixture of liquids, gases and microorganisms), fluid flowunit operation 104 (e.g., pump or other device capable of causing afluid to move), loop section 106 and first non-vertical pressurereduction zone 108 can be a metallic, non-metallic, or compositestructure. For example, the elements can include one or more metallicmaterials such as 304, 304L, 316, or 316L stainless steels. In someinstances, one or more coatings, layers, overlays, inserts, or othermaterials can be deposited on, applied to, joined with, or formedintegral to all or a portion of the metallic, non-metallic or compositestructures to beneficially or detrimentally affect the ability formicrobiological organisms to attach thereto or to grow thereupon. Forexample, a coating inhibiting the growth or attachment ofmicrobiological organisms may be deposited on or formed integral withthe surfaces of the loop reactor 101 that are thermally conductivelycoupled to heat transfer unit operation 116. In another example, acoating that inhibits the growth or attachment of biological organismsmay be deposited on or formed integral with portions of loop reactor 101where it is desired to achieve removal of accumulated biomass moreeasily.

In at least some instances, the construction of elements of loop reactor101 can include features that facilitate sterilization of all or aportion of the process contact surfaces. Such sterilization can beaccomplished for example using steam sterilization, ultravioletsterilization, chemical sterilization, or combinations thereof. In atleast some instances, one or more non-metallic materials or one or morenon-metallic coatings may be used within all or a portion of theinterior or exterior of some or all of the elements of loop reactor 101.The use of such non-metallic materials may advantageously provide, forexample, sterializable surfaces that are capable of supporting orpromoting biological growth.

Gas/liquid separation vessel 102 can include any number of devices,systems, or combinations thereof to separate the multi-phase mixture 121into at least a gas effluent 123 and a liquid effluent 125 which operateon the same principles as gas/liquid separators used with conventionalbioreactors. In at least some instances, biosolids present in themulti-phase mixture 121 may be separated into a solids-containingeffluent. In at least some instances, at least a portion of thesolids-containing effluent from the gas/liquid separation vessel 102 canbe combined with the one or more liquids and the mixture returned togas/liquid separation vessel or the loop section 106. In at least someinstances, the gas/liquid separation vessel 102 can include one or moregas/liquid separators operating in parallel or series.

The gas/liquid separation vessel 102 can include one or more passiveseparators (e.g., one or more wet cyclones or the like) capable ofseparating the gas effluent 123 and the liquid effluent 125 from themulti-phase mixture 121. In at least some instances, the passiveseparator may also include a solids separation section to separate atleast a portion of the biosolids present in the multi-phase mixture 121.In other instances, the gas/liquid separation vessel 102 can include oneor more active separation devices (e.g., a three-phase rotary separator)capable of separating the gas effluent 123, the liquid effluent 125, andthe solids-containing effluent from the multi-phase mixture 121.

In at least some instances, the gas effluent 123 may include a mixtureof one or more gas substrates (e.g., methane or carbon monoxide) and oneor more gaseous byproducts (e.g., carbon dioxide) generated as abyproduct by the biological organisms in the loop reactor 101. In atleast some instances, the gas effluent 123 may be separated and at leasta portion of the one or more gas substrates recycled (not shown) to theloop reactor 101, for example as a gas substrate. In at least someinstances, the gas effluent 123 may include one or more usefulcompounds. For example, the gas effluent 123 may contain an amount ofone or more gaseous C₂₊ hydrocarbon compounds and compounds basedthereupon having value as either a finished product or as a raw materialin a subsequent process. Such useful compounds may be separated from thegas effluent 123 prior to recycling at least a portion of the gaseffluent 123 to the loop reactor 101.

In at least some instances, the liquid effluent 125 will include amixture containing one or more liquids, nutrients, and the likeintroduced to the loop reactor 101 by nutrient and/or mineral supplysubsystem 114. In at least some instances, the liquid effluent 125 maybe removed from the loop reactor and returned to the gas/liquidseparation vessel 102 by spraying onto the surface of the multi-phasemixture in the gas/liquid separation vessel 102 in order to reducefoaming within gas/liquid separation vessel 102. Anti-foam agents may beadded to the liquid effluent 125 sprayed into the gas/liquid separationvessel 102 or maybe sprayed into the gas/liquid separation vessel 102without the liquid effluent 125. In at least some instances, the liquideffluent 125 may include one or more useful compounds. For example, theliquid effluent 125 may contain an amount of one or more liquid C₂₊hydrocarbon compounds including, but not, limited to alcohols, ketones,glycols, and other compounds based thereupon having value as either afinished product or as a raw material in a subsequent process. Suchuseful hydrocarbon compounds may be separated from the liquid effluent125.

In some instances, the reactor is used to produce natural or non-naturalproducts, such as ethanol, acetate, butanol, isoprene, propylene,isoprene, enzymes, or other metabolites or cellular products wherein theproduct is derived from a microorganism. In such cases, the products maybe present in either the gas effluent 123 or the liquid effluent 125depending on the physical properties of the product.

In at least some instances, the bottom of gas/liquid separation vessel102 can be shaped, formed, or configured to promote the accumulation ofbiological material 127 (i.e., “biosolids” or “biomass”) at a desiredlocation within vessel 102. For example, the bottom of gas/liquidseparation vessel 102 can be conically shaped, dished, or sloped suchthat biosolids 127 settling to the bottom of vessel 102 preferentiallycollect in one or more predetermined locations. In the embodimentillustrated in FIG. 3, liquid effluent 125 and biosolids 127 can beremoved from the bottom of gas/liquid separation vessel 102 anddelivered to fluid flow unit operation 104, e.g., a pump. The liquideffluent 125 and biosolids 127 removed from gas/liquid separation vessel102 can be received at inlet 129 of pump 104 and output from an outlet131 of pump 104. Outlet 131 of pump 104 is in fluid communication withinlet 133 of loop section 106 of loop reactor 101. Suitable pumps formoving liquid effluent 125 and biosolids 127 include pumps capable ofmoving fluids (liquids or gases) and slurries, by mechanical action andwhich are able to produce desired flow rates in the substantial absenceof shear forces detrimental to the biomass and/or cavitation. Avoidingcavitation is desirable because cavitation causes gaseous substrates andnutrients in the multi-phase mixture to come out of solution making themless accessible to the biomass. Examples of such type of pumps arecentrifugal pumps, although pumps which are not centrifugal pumps mayalso be used. For example, positive displacement pumps, progressivecavity pumps, double diaphragm pumps, and the like can also be used.Devices other than pumps can also be used to move the multi-phasemixture, for example, propellers driven by a motor, such as thepropellers and motors described in U.S. Pat. No. 7,579,163 can be usedinstead of or in combination with a pump.

In FIGS. 3 and 4, outlet 131 of fluid flow unit operation 104 is influid communication with an inlet 133 of loop section 106. Loop section106 extends from its inlet 133 to an outlet 135 of loop section 106.Outlet 135 of loop section 106 is in fluid communication with gas/liquidseparation vessel 102. Loop section 106 can be formed from piping madefrom materials that do not adversely affect reaction/fermentationprocesses carried out using loop reactor 101. For example, a loopsection 106 can be formed from piping made from the materials describedabove for elements of loop reactor 101. The cross-sectional area of loopsection 106 may be constant or the loop section 106 may include one ormore sections that have different cross-sectional areas. Reference tothe cross-sectional area of loop section 106 in the present disclosuredoes not include the cross-sectional area of gas/liquid separationvessel 102. The inner diameter of the loop section 106 may vary over awide range. Exemplary diameters range from about 20 centimeters to 3meters. Other exemplary diameters range from 25 centimeters to 2.5meters. When loop section 106 includes sections of differingcross-sectional areas, the sections of loop section 106 having largercross-sectional area have cross-sectional areas that are at most threetimes the cross-sectional area of the sections of loop section 106having smaller cross-sectional areas. In other exemplary embodiments,sections of loop section 106 having larger cross-sectional area, havecross-sectional areas that are at most two times the cross-sectionalarea of the sections of loop section 106 having smaller cross-sectionalareas. In yet other exemplary embodiments, sections of loop section 106having larger cross-sectional area, have cross-sectional areas that areat most 0.5 times the cross-sectional area of sections of loop section106 having smaller cross-sectional areas. The length of loop section 106can vary depending upon a number of factors, including the desiredlength of time the multi-phase mixture 121 resides in loop section 106.The length of loop section 106 may also be determined based on otherfactors such as, but not limited to total reactor/liquid volume desired,total pressure drop across the loop, desired substrate utilization andyield. In exemplary embodiments, loop section 106 can vary in the lengthat its centerline from about 30 m to about 250 m, 40 m to about 200 m,50 m to about 150 m and 60 to about 100 m.

The embodiments of loop section 106 illustrated in FIGS. 3 and 4 areU-shaped, including two elbows 137 that bend at 90° angles when viewedfrom above. Loop section 106 can take other shapes. For example, loopsection 106 can include more than the two 90° elbows 137 or it caninclude more than one elbow that is less than 90°. In other embodiments,loop section 106 can include numerous elbows that are greater than 90°or less than 90°.

Outlet 135 of loop section 106 is elevated relative to inlet 133 of loopsection 106. Loop section 106 accommodates for this difference inelevation between its inlet 133 and its outlet 135 by being sloped. Thespecific slope of the loop section 106 or portions of loop section 106depend in part on the length of loop section 106, the vertical distancebetween the centerline of loop section 106 at its inlet 133 and thecenterline of loop section 106 at its outlet 135, and whether loopsection 106 includes a second pressure reduction zone 112 that is nothorizontal. Loop section 106 can be sloped upward from its inlet 133 toits outlet 135 to accommodate for the change in elevation between inlet133 and outlet 135. Alternatively, a portion of loop section 106 can besloped downward and a portion of loop section 106 can be sloped upward.In such alternative embodiments, the portion of loop section 106 that issloped upward accounts for the loss in elevation resulting from thepresence of the downward sloped portion of loop section 106 and thedifference in elevation between inlet 133 of loop section 106 and outlet135 of loop section 106. For example, the portion of loop section 106extending from its inlet 133 to the first 90° elbow 137 in FIGS. 3 and 4can be sloped downward, and the portion of loop section 106 extendingfrom the first or second elbow 137 can be sloped upward to outlet 135 ofloop section 106.

In embodiments of loop reactor 101 that include a second pressurereduction zone 112 which is not horizontal and accounts for a portion ofthe elevation change from exit 131 of fluid flow unit operation 104 tooutlet 135 of loop section 106, the amount of elevation change that mustbe provided by the balance of non-vertical loop section 106 (i.e., theportion of loop section 106 that is not vertical) is reduced. When asecond pressure reduction zone 112 which accounts for a portion of theelevation change from exit 131 of fluid flow unit operation 104 tooutlet 135 of loop section 106 is not present, the amount of theelevation change provided by the balance of the non-vertical loopsection 106 is greater compared to when such second pressure reductionzone 112 is present. In exemplary embodiments of loop reactor 101described herein, which include a second pressure reduction zone 112which accounts for a portion of the elevation change from exit 131 offluid flow unit operation 104 to outlet 135 of loop section 106, suchsecond pressure reduction zone 112 accounts for no more than 90% of theelevation change from the centerline of loop section 106 at its inlet133 to outlet 135 of loop section 106, for no more than 80% of theelevation change from the centerline of loop section 106 at its inlet133 to outlet 135 of loop section 106, for no more than 70% of theelevation change from the centerline of loop section 106 at its inlet133 to outlet 135 of loop section 106, for no more than 60% of theelevation change from the centerline of loop section 106 at its inlet133 to outlet 135 of loop section 106, for no more than 50% of theelevation change from the centerline of loop section 106 at its inlet133 to outlet 135 of loop section 106, no more than 40% of the elevationchange from the centerline of loop section 106 at its inlet 133 tooutlet 135 of loop section 106, no more than 30% of the elevation changethe centerline of loop section 106 at its inlet 133 to outlet 135 ofloop section 106, no more than 20% of the elevation change from thecenterline of loop section 106 at its inlet 133 to outlet 135 of loopsection 106, no more than 10% of the elevation change from thecenterline of loop section 106 at its inlet 133 to outlet 135 of loopsection 106 or no more than 5% of the elevation change from thecenterline of loop section 106 at its inlet 133 to outlet 135 of loopsection 106.

The exemplary embodiments illustrated in FIGS. 3 and 4 include aplurality of static mixers 139, positioned along the length of loopsection 106. Benefits of the use of static mixers are described in U.S.Pat. No. 7,579,163 and include mixing of the nutrient gases into themulti-phase mixture. Exemplary types of static mixers are also describedin the '163 patent. Static mixers that can be used in embodimentsdescribed are not limited to those described in the '163 patent. Staticmixers other than those described in the '163 patent can be used in theembodiments described herein. For example, other types of static mixersare available from companies such as StaMixCo LLC of Brooklyn, N.Y. andSulzer Management Ltd. of Winterthur, Switzerland. In the exemplaryembodiment illustrated in FIGS. 3 and 4, 50 static mixers 139 areschematically represented by 23 blocks. The static mixers 139 of theexemplary embodiment of FIGS. 3 and 4 can be provided at a density ofabout one mixer per three meters of the loop section 106 when the staticmixer has a length of about 1 meter. In other words, in certaininstances, static mixers are spaced apart by a distance about equal to 3times the length of one of the static mixers. The number of staticmixers is not limited to 50 nor is their density limited to one mixerper 3 meters of loop section 106. In accordance with embodimentsdescribed herein, fewer or greater numbers of static mixers can beprovided and the static mixers may be provided at a lesser or greaterdensity. The particular number of static mixers used and the density atwhich they are deployed will be determined in part based upon theircontribution to mass transfer of gas into the liquid and microorganismsand/or the pressure drop produced by the static mixers.

Continuing to refer to FIGS. 3 and 4, in exemplary embodiments, system100 includes a nutrient and/or mineral supply subsystem 114 forintroducing nutrients and minerals into loop section 106 at one or morelocations between the outlet 131 of fluid flow unit operation 104 andfirst non-vertical pressure reduction zone 108. Introducing nutrientsand/or minerals upstream of the first non-vertical pressure reductionzone 104 results in the introduced nutrients and/or minerals beingpresent in portions of the loop section where the microorganisms aremore active and the demand for the nutrients and/or minerals is high.Compared to portions of the loop section upstream of the firstnon-vertical pressure reduction zone, the microorganisms activitydownstream of the first non-vertical pressure reduction zone 104 islower, thus making introduction of the nutrients and/or minerals betweenthe first non-vertical pressure reduction zone 104 and gas/liquidseparation vessel 102 less effective. Such nutrients include nutrientscapable of supporting or transporting dissolved or suspended sustenanceto biomass forming microbiological organisms in the multi-phase mixturewithin the loop reactor 101. In the embodiment illustrated in FIGS. 3and 4, nutrients and minerals are introduced at two locations along loopsection 106 between the outlet 131 of fluid flow unit operation 104 andfirst non-vertical pressure reduction zone 108; however, in accordancewith other embodiments, nutrient and/or mineral supply subsystem 114 canintroduce nutrients and minerals at different locations along loopsection 106 and can introduce nutrients/minerals at fewer than twolocations or more than two locations along loop section 106. Subsystem114 provides gaseous substrates/nutrients for introduction into a liquidculture medium to form a multi-phase mixture of the liquid culturemedium and supplied gaseous substrates/nutrients. Such gaseoussubstrates/nutrients can include a single gas or a combination of gasescapable of supporting or providing sustenance or nutrients to thebiomass producing biological organisms in the loop reactor 101. Asillustrated in FIGS. 3 and 4, exemplary nutrients include natural gas,nitrogen, oxygen and ammonia water. A source of steam can be providedfor thermal energy and cleaning purposes. Nutrients that can be suppliedby nutrient subsystem 114 are not limited to natural gas, nitrogen,oxygen and ammonium water. Other nutrients/minerals, such as methane,syngas, water, phosphate (e.g., as phosphoric acid), nitrates, urea,magnesium, calcium, potassium, iron, copper, zinc, manganese, nickel,cobalt and molybdenum, typically used as sulfates, chlorides or nitratescan also be provided by nutrient subsystem 114.

In exemplary embodiments, system 100 includes a heat transfer unitoperation 116 for introducing or removing thermal energy from themulti-phase mixture in loop section 106. Heat transfer unit operation116 can introduce thermal energy to or remove thermal energy from themulti-phase mixture in the loop section 106 at one or more locationsalong loop section 106. In the embodiments illustrated in FIGS. 3 and 4,heat transfer unit operation 116 removes or introduces thermal energy atone location along loop section 106; however, thermal energy can beremoved or introduced at more than one location along loop section 106.In at least some instances, the microbiological activity that occurswithin the loop reactor 101 generates heat as a byproduct. Leftuncontrolled, such heat can adversely affect the metabolism or health ofthe microbiological organisms within the loop reactor 101.Alternatively, microbiological organisms may also have a temperaturebelow which the metabolism or health of the organism is adverselyaffected. As such, the biological organisms within the loop reactor 101have a defined temperature range providing optimal growth and metabolicconditions. In at least some instances, the multi-phase mixture withinthe loop reactor 101 can be maintained at a temperature of about 130° F.or less; about 120° F. or less; about 110° F. or less; about 100° F. orless; about 95° F. or less; about 90° F. or less; about 85° F. or less;or about 80° F. or less using the heat transfer unit operation 116. Inat least some instances, the multi-phase mixture within the loop reactor101 can be maintained at a temperature of from about 55° F. to about120° F.; about 60° F. to about 110° F.; about 110° F. to about 120° F.;about 100° F. to about 120° F.; about 65° F. to about 100° F.; about 65°F. to about 95° F.; or about 70° F. to about 90° F. using heat transferunit operation 116.

In exemplary embodiments described herein, gas pressure in headspace 143of gas/liquid separation unit operation 102 ranges from about 0.2 toabout 0.6 bars gauge; however, the gas pressure in the headspace 143 isnot limited to a range of about 0.2 to about 0.6 bars gauge. Forexample, in exemplary embodiments described herein, the gas pressure inheadspace 143 can be less than 0.2 bars or greater than about 0.6 barsgauge. The pressure at outlet 131 of pump 104 ranges from about 2.5 barsto about 4.0 bars gauge; however, the pressure at outlet 131, of pump104 is not limited to a range of about 2.5 bars to about 4.0 bars gauge.For example, in exemplary embodiments described herein, the pressure atoutlet 131 of pump 104 can be less than about 2.5 bars or greater thanabout 4.0 bars gauge. In exemplary embodiments that include staticmixers 139, the pressure drop across a static mixer ranges from about0.03 to about 0.05 bars gauge; however, the pressure drop across astatic mixer is not limited to a range from about 0.03 to about 0.05bars gauge. For example, in exemplary embodiments described herein, thepressure drop across a static mixer may be less than 0.03 bars orgreater than 0.05 bars gauge. In accordance with exemplary embodimentsdescribed herein, pressure within loop section 106 at the beginning ofnon-vertical pressure reduction zone 108 ranges from about 1.5 to about2.5 bars gauge; however, the pressure within loop section 106 at thebeginning of non-vertical pressure reduction zone 108 is not limited toa range from about 1.5 to about 2.5 bars gauge. For example, pressurewithin loop section 106 at the beginning of non-vertical pressurereduction zone 108 may be less than about 1.5 bars or greater than about2.5 bars. In accordance with exemplary embodiments described herein,pressure within loop section 106 at the end of non-vertical pressurereduction zone 108 ranges from about 0.2 bars to about 0.6 bars gauge;however, the pressure within loop section 106 at the end of non-verticalpressure reduction zone 108 is not limited a range of about 0.2 bars toabout 0.6 bars gauge. For example, in accordance with embodimentsdescribed herein, pressure within loop section 106 at the end ofnon-vertical pressure reduction zone 108 can be less than about 0.2 barsor greater than about 0.6 bars gauge. In embodiments described herein,the pressure drop across non-vertical pressure reduction zone 108 canrange from about 1.2 bars to about 2.3 bars gauge; however, the pressuredrop across the non-vertical pressure reduction zone 108 is not limitedto a range from about 1.2 bars to about 2.3 bars gauge. For example, thepressure drop across the non-vertical pressure reduction zone 108 can beless than 1.2 bars or more than 2.3 bars gauge. In some instances, thepressure drop across non-vertical pressure reduction zone 108 accountsfor at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70% or at least 80% of the pressure drop between theoutlet of fluid flow unit operation 104 and the headspace 143 ofgas/liquid separation vessel 102. The foregoing description regardingpressure drops across non-vertical pressure reduction zone 108 andpercentage of the pressure drop between the outlet of fluid flow unitoperation 104 and headspace 143 of gas/liquid separation vessel 102attributable to non-vertical pressure reduction zone 108 applies equallyto the pressure drop across pressure reduction device 145 that islocated in pressure reduction zone 108.

In embodiments illustrated in FIGS. 3 and 4, first non-vertical pressurereduction zone 108 is located downstream of the last static mixer 139and upstream of the outlet 135 of loop section 106 which is in fluidcommunication with gas/liquid separation unit operation 102. Firstnon-vertical pressure reduction zone 108 includes a pressure reductiondevice 145. In accordance with embodiments illustrated in FIGS. 3 and 4,the pressure within loop section 106 immediately downstream of pressurereduction device 145 is less than the pressure within loop section 106immediately upstream of pressure reduction device 145. Pressurereduction device 145 causes the pressure within loop section 106immediately downstream of pressure reduction device 145 to be less thanthe pressure within loop section 106 immediately upstream of pressurereduction device 145. Preferred devices for use as pressure reductiondevice 145 include devices that provide the desired reduction inpressure by means other than a change in hydrostatic pressure andwithout exposing the liquid culture media and microorganisms containedtherein to forces resulting from shearing or cavitation that damage themicroorganisms. For example, pressure reduction device 145 can be a flowcontrol device, such as a control valve or a back pressure control valve(as opposed to a check valve) or an expansion joint (e.g., a pipe jointhaving an upstream diameter that is less than its downstream diameter)or a combination of multiple expansion joints or a combination of acontrol valve and one or more expansion joints. Exemplary control valvesinclude control valves that are actuated hydraulically, pneumatically,manually, by a solenoid, or by a motor; however, control valves, usefulin embodiments described herein, are not limited to the foregoing typesof control valves. Likewise, pressure reduction device 145 is notlimited to control valves and expansion joints and combinations thereof.For example, pressure reduction device 145 can be a device that is not acontrol valve or an expansion joint that results in the pressure withinloop section 106 immediately downstream of the device being less thanthe pressure within loop section 106 immediately upstream of the device.

In accordance with embodiments described herein, pressure reductiondevice 145 can be a variable pressure reduction device, such as acontrol valve which can control media flow by varying the size of theflow passage, e.g., manually or based on a signal from a controller thatis implementing a feedback control loop based on input from sensorswhich detect process parameters, such as pressure, temperature, gasconcentration (e.g., oxygen, carbon dioxide, methane) pH, liquid mediadensity circulation rate, biomass concentrations, or flow times betweentwo points along the loop section 106. Employing a variable pressurereduction device allows for the difference in the pressure within loopsection 106 immediately upstream of the device and the pressure withinloop section 106 immediately downstream of the device to be adjusted byvarying the degree to which the device is open. For example, thedifference in the pressure can be decreased by opening the device andthe difference pressure can be increased by closing the device. Theability to vary the pressure within the loop section 106 upstream of thevariable pressure reduction device provides operators the ability tobetter control the processes occurring within loop section 106. Forexample, the variable pressure reduction device can be used to decreasethe pressure within loop section 106 upstream of the variable pressurereduction device by opening (increasing the flow rate through) thevariable pressure reduction device. Reducing the pressure within loopsection 106 allows operators to slow down mass transfer, reduceproduction rates, reduce nutrient demands and increase rates of gasdesorption from the multi-phase mixture. The variable pressure reductiondevice can be used to increase the pressure within loop section 106upstream of the variable pressure reduction device by closing (reducingthe flow rate through) the variable pressure reduction device.Increasing the pressure in loop section 106 allows operators to increasethe mass transfer rate, increase production rate, increase nutrientdemands and decrease rates of gas desorption from the multi-phasemixture.

Utilizing a variable pressure reduction device also provides operatorswith the ability to better control the pressure within loop section 106downstream of the variable pressure reduction device. For example,utilizing the variable pressure reduction device to decrease thepressure within loop section 106 downstream of the variable pressurereduction device allows operators to promote the desorption of gases(e.g., carbon dioxide) which can inhibit the biological processoccurring in the loop section. Utilizing the variable pressure reductiondevice to increase the pressure within loop section 106 downstream ofthe variable pressure reduction device allows operators to inhibit thedesorption of gases (e.g., nutrient gases such as oxygen and methane)which are needed to fuel the biological processes occurring in the loopsection 106. Inhibiting the desorption of gases such as oxygen andmethane may be desired in order to manage the risk of combustion fueledby the oxygen and methane gas.

Referring to FIG. 7D, an exemplary variable pressure reduction device145 useful in the non-vertical pressure reduction zone 108 of loopreactors 100 in accordance with embodiments described herein isillustrated. One end of variable pressure reduction device 145 isattached to a portion of loop section 106 that is upstream of variablepressure reduction device 145. The other end of variable pressurereduction device 145 is attached to the portion of loop section 106 thatis downstream of variable pressure reduction device 145. Referring toFIG. 7A, variable pressure reduction device 145 includes an eccentricreducer 701. Eccentric reducer 701 includes a pipe section 703 having asubstantially constant inner diameter and an eccentric reducer section705. The inner diameter of pipe section 703 is constant and issubstantially the same as the inner diameter of the portion of loopsection 106 to which pipe section 703 is attached. Eccentric reducersection 705 includes an end adjacent pipe section 703 which has an innerdiameter that is equivalent to the inner diameter of pipe section 703.The smaller end of eccentric reducer 705 opposite the end adjacent topipe section 703 has a smaller diameter. The diameter of the smaller endof eccentric pipe reducer 705 is equivalent to the diameter of thecontrol valve 711 described below which is downstream of eccentric pipereducer 705. Between the two ends of the eccentric reducer 705, theinner diameter transitions from the larger diameter end to the smallerdiameter end and has an edge that is parallel to the portion of loopsection 106 to which it is connected and the portion of the controlvalve 711 to which it is connected.

Referring to FIGS. 7A, 7B and 7D, variable pressure reduction device 145includes a control valve 711 attached to the smaller end of eccentricreducer 701. Control valve 711 has an inner diameter that issubstantially equivalent to the inner diameter of the smaller end ofeccentric pipe reducer 705. Media flow through control valve 711 can beadjusted by varying the size of the flow passage within control valve711 by manipulation of the handle 713 of control valve 711. As describedabove, handle 713 can be manipulated by an electronic controller.

Referring to FIGS. 7A, 7C and 7D, the end of variable pressure reductiondevice 145 opposite eccentric reducer 701 includes an eccentric expander719. Eccentric expander 719 includes a pipe section 721 having asubstantially constant inner diameter and an eccentric expander section723. The inner diameter of pipe section 721 is constant and issubstantially the same as the inner diameter of control valve 711 towhich pipe section 721 is attached. Eccentric expander section 723includes an end adjacent to pipe section 721 which has an inner diameterthat is equivalent to the inner diameter of pipe section 721. The largerend of eccentric expander 723 opposite the end attached to pipe section721 has an inner diameter that is substantially equivalent to the innerdiameter of the portion of loop section 106 that is attached to thelarger end of eccentric expander 723. In some embodiments, the innerdiameter of the loop section 106 downstream of variable pressurereduction device 145 and the inner diameter of the end of eccentricexpander 723 attached to a portion of loop section 106 that isdownstream of eccentric expander 723 are larger than the diameter of theloop section 106 upstream of pressure reduction device 145. Between thetwo ends of the eccentric expander 719, the inner diameter transitionsfrom the smaller diameter end to the larger diameter end and has an edgethat is parallel to the portions of loop section 106 to which it isconnected and an edge of the control valve 711 to which it is connected.

In accordance with other embodiments of variable pressure reductiondevice 145, one or both of eccentric reducer 701 and eccentric expander723 are omitted. In such embodiments, one end of control valve 711 isattached to an end of loop section 106 that is upstream from controlvalve 711 and the other end of control valve 711 is attached to in aloop section 106 that is downstream of control valve 711. Utilization ofeccentric reducer 701 and eccentric expander 723 facilitate utilizationof a control valve having an inner diameter that is smaller than theinner diameter of a control valve that would be needed if the eccentricreducer 701 and the eccentric expander 723 are not utilized. A controlvalve with a smaller inner diameter (compared to a similar control valvewith a larger inner diameter) is able to control the pressure dropacross the valve with more precision and greater sensitivity. Suchprecision and greater sensitivity may be preferred in someimplementations.

Alternatives to a control valve 713 for use in pressure reduction device145 include one or more expansion joints or concentric expanders whichcause the pressure in loop section 106 downstream of the expansionjoint/concentric expander to be reduced compared to the pressure in theloop section 106 upstream of the expansion joint/concentric expander.

In accordance with embodiments described herein and illustrated in FIG.3, downstream of the first pressure reduction zone 108, loop section 106can include a second pressure reduction zone 112. In embodimentsillustrated in FIG. 3, second pressure reduction zone 112 is locateddownstream of the first pressure reduction zone 108 and upstream of theoutlet 135 of loop section 106, which is in fluid communication withgas/liquid separation unit operation 102.

In the embodiment illustrated in FIG. 3, second pressure reduction zone112 is provided by modifying loop section 106 to include a section thatis oriented vertically. The vertical orientation of a section of loopsection 106 provides a second pressure reduction zone 112 that resultsin the pressure within loop section 106 at the upper end of secondpressure reduction zone 112 being less than the pressure within loopsection 106 at the lower end of the second pressure reduction zone 112.The pressure reduction provided by second pressure reduction zone 112 isattributable, at least in part, to the difference in hydrostaticpressure from the top to the bottom of the second pressure reductionzone 112. The length of the vertical portion of second pressurereduction zone 112 can be determined at least in part based upon thedesired reduction in pressure to be provided by second pressurereduction zone 112. For example, in exemplary embodiments, the length ofthe vertical portion of second pressure reduction zone 112 ranges fromabout 1 meter to less than about 10 meters; however, the length of thevertical portion of second pressure reduction zone 112 is not limited toa range from about 1 meter to less than about 10 meters. For example,the length of the vertical portion of the second pressure reductiondevice can be less than about 1 meter or greater than about 10 meters.Second pressure reduction zone 112 can also include a pressure reductiondevice 147 of the type described above with respect to first pressurereduction device 145. Utilizing a second pressure reduction zone 112provides added flexibility in controlling the pressure within loopsection 106 which can lead to greater precision in controlling thepressure which can lead to an improved process productivity andstability. In certain embodiments, the second pressure reduction zone112 accounts for 60% or less, 50% or less, 40% or less, 30% or less, 20%or less or 10% or less of the length of the vertical distance betweenthe gas/liquid interface 118 in gas/liquid separation unit operation 102and the centerline of loop section 106 at outlet 131 of fluid flow unitoperation 104.

Referring to the embodiments of FIG. 4, an optional second pressurereduction zone 113 can include a pressure reduction device of the typedescribed above with respect to pressure reduction device 145. Inaccordance with embodiments of FIG. 4, second pressure reduction zone113 is a non-vertical pressure reduction zone and includes a pressurereduction device. In exemplary embodiments, first pressure reductiondevice 145 of the first pressure reduction zone 108 is separated fromthe pressure reduction device of the second pressure reduction zone 113by a non-vertical portion of the loop section 106. In accordance withembodiments illustrated in FIG. 4, the multi-phase mixture in loopsection 106 flows from first non-vertical pressure reduction zone 108 tothe gas/liquid separation unit operation 102 without flowing in avertical direction. In accordance with embodiments according to FIG. 4,when a second pressure reduction zone 113 is present, it accounts forless of a pressure drop compared to the pressure drop across firstpressure reduction zone 108. For example, the pressure drop acrosssecond pressure reduction zone 113 is about equal to the pressuredifference between headspace 143 of gas/liquid separation vessel 102 andthe pressure at the outlet of the first pressure reduction zone 108and/or pressure reduction device 145. Such pressure drop across secondpressure reduction zone 113 can range between about 0.1 bars to about0.5 bars; however, the pressure drop across second pressure reductionzone 113 is not limited to a range between about 0.1 bars to about 0.5bars. For example the pressure drop across second pressure reductionzone 113 can be less than 0.1 bars or greater than 0.5 bars. In someinstances, the pressure drop across second pressure reduction zone 113accounts for less than 10%, less than 5%, less than 3% or less than 2%of the pressure drop from the outlet of fluid flow unit operation 104 tothe headspace 143 of gas/liquid separation vessel 102. The foregoingdescription regarding pressure drops across second pressure reductionzone 113 and percentage of the pressure drop from the outlet of fluidflow unit operation 104 to headspace 143 of gas/liquid separation vessel102 attributable to pressure reduction zone 113 applies equally to thepressure drop across second pressure reduction device 147 in pressurereduction zone 112 of FIG. 3. Utilizing a second pressure reduction zone113 provides added flexibility in controlling the pressure within loopsection 106 which can lead to greater precision in controlling thepressure which can lead to improved process productivity and stability.

Loop section 106 upstream of first non-vertical pressure reduction zone108 includes a desorption gas inlet 149. In the illustrated embodiment,desorption gas inlet 149 is in fluid communication with a source ofdesorption gas, e.g., nitrogen, and in fluid communication with anon-vertical section of loop section 106. Thus, in accordance withembodiments illustrated in FIGS. 3 and 4, desorption gas can beintroduced into a non-vertical section of loop section 106. Introducinga desorption gas into the multi-phase mixture at desorption gas inlet149 causes a decrease in the partial pressure of other gases present inthe multi-phase mixture (e.g., carbon dioxide and methane). Reducing thepartial pressure of other gases present in the multi-phase mixture canhave the effect of reducing the mass transfer of nutrient gases into themicroorganism and/or causing the other gases to come out of solution.

In an alternative embodiment, the desorption gas inlet 149 is located ina non-vertical section of loop section 106 between first pressurereduction zone 108 and outlet 135 of loop section 106. Providing thedesorption gas inlet 149 at this location allows for the introduction ofthe desorption gas in a section of loop section 106 downstream of thefirst pressure reduction zone where the pressure has been reduced bypassing the multiphase mixture through the first pressure reduction zone108 and/or the second pressure reduction zone 112 in FIG. 3 or 113 inFIG. 4. As described in the previous paragraph, introduction of adesorption gas into the multi-phase mixture causes a decrease in thepartial pressure of other gases present in the multi-phase mixture(e.g., carbon dioxide and methane). Reducing the partial pressure ofother gases present in the multi-phase mixture can have the effect ofreducing the mass transfer of nutrient gases into the microorganismand/or causing the other gases to come out of solution. Locating thedesorption gas inlet 149 downstream of first pressure reduction zone108, avoids introducing the desorption gas into the multi-phase mixtureat a location where the desorption gas can affect the performance of thefirst pressure reduction zone 108 and/or second pressure reduction zones112 or 113. For example, gas that separates from the multi-phase mixturecan affect the performance of the first pressure reduction zone 108 inreducing the pressure. For example, if the first pressure reduction zone108 includes a pressure reduction device in the form of a control valve,increasing amounts of gas desorbed from the multi-phase mixture can makeit more difficult for the valve to control flow and pressure reduction.Introducing the desorption gas downstream of the first pressurereduction zone 108 avoids this problem.

FIG. 5 shows a high level method of operation 500 of a system 100 forstimulating production of biomass using one or more loop reactors 101described in detail above with regard to FIGS. 2-4. Such systemsadvantageously introduce one or more gaseous substrates and a liquidmedia containing one or more nutrients into a liquid culture mediacontaining at least one microorganism capable of utilizing the gaseoussubstrates and liquid nutrients to grow. The combination of the one ormore gaseous substrates, liquid media containing one or more nutrientsand liquid culture media containing at least one microorganism resultsin a multi-phase mixture that is circulated through a loop reactor 101.The conditions within the loop reactor 101 are controlled to promotemass transfer and subsequent microbiological uptake of the gaseoussubstrate and liquid nutrients, reduction of pressure within the loopreactor and desorption of gases from the multi-phase mixture. Themulti-phase mixture after passing through the loop section 106 of theloop reactor 101 is received by a gas/liquid separation unit operation102 where the multi-phase mixture is separated into liquid and gasphases. The method commences at 502.

At 504 a gaseous substrate is dispersed within the liquid media to formthe multi-phase mixture. Such dispersion may occur at or near inlet 133of loop section 106, although additional quantities of gaseous substratemay be introduced into the liquid culture media at other locations ofloop section 106 and the liquid media at or near the inlet 133 of loopsection 106 may already contain some dissolved gaseous substrates. Insome instances, gaseous substrate may be dispersed at multiple pointsalong loop section 106 and the gaseous substrate at each dispersionpoint may have the same or a different temperature, pressure,composition, or combinations thereof. The ability to vary physical orcompositional properties of the gaseous substrate at different locationsalong the loop section 106 advantageously permits the tailoring of thegaseous substrate not only to the specific microbiological speciespresent in the multi-phase mixture, but also to the specific location ofthe microbiological species within the loop section 106 based on thedispersion point of the gaseous substrate.

At 506 the multi-phase mixture is flowed through the loop section 106 ofloop reactor 101. As the multi-phase mixture flows through the loopsection 106, it contacts a plurality of static mixers 139, which promotethe mixing of the gaseous substrate and/or nutrients into the liquidculture medium. By adjusting or otherwise controlling the flow rate ofthe multi-phase mixture through loop reactor 101, the length of time thebubbles of gaseous substrate and nutrients are in contact with themicroorganism(s) can be modified. Increasing the length of time thebubbles of gaseous substrate and nutrients are in contact with themicroorganism(s) can increase the amount of mass transfer of gaseousmaterials into the microorganisms and the microbiological uptake ofgaseous materials by the microorganism. Conversely, decreasing thelength of time the bubbles of gaseous substrate and nutrients are incontact with the microorganism(s) can decrease the amount of masstransfer of gaseous materials into the microorganisms and themicrobiological uptake of gaseous materials by the microorganisms. Insome instances, the length of time the bubbles of the gaseous substrateand nutrients are in contact with the microorganisms can be measured andcontrolled. For example, a control subsystem 290 can alter, adjust orcontrol the fluid velocity of the multi-phase mixture through the loopreactor. In some instances, the temperature, pressure, or composition ofthe gaseous substrate may be altered, adjusted or controlled via thecontrol subsystem 290 to maintain a desired gas substrate bubble sizewithin loop reactor 106. In other instances, the temperature, pressure,or composition of the gas substrate may be altered, adjusted orcontrolled via the control subsystem 290 to maintain the concentrationof one or more gas substrate components (e.g., methane, carbon dioxide,hydrogen, oxygen, nitrogen, etc.) within the liquid phase of themulti-phase mixture.

At 508 the temperature of the multi-phase mixture within loop reactor101 can be altered, adjusted, or controlled to maintain the temperaturewithin a defined temperature range. In at least some instances, thedefined temperature range may be selected or otherwise chosen based atleast in part on the microbiological species used within system 100.Excess heat may be generated as a byproduct by the microbiologicalorganisms responsible for at least a portion of the activity withinsystem 100. This excess heat, if left uncontrolled, could inhibit oradversely affect the growth or metabolism of some or all of themicrobiological organisms within system 100. In at least some instances,cooling of the multi-phase mixture in loop reactor 101 may be providedto maintain the temperature of the multi-phase mixture in loop reactor101 within a defined range. Such cooling may include passage of acooling media through reservoirs or coils thermally conductively coupledto the loop reactor 101 or a conduit which has diverted a portion of themulti-phase mixture out of the loop reactor 101 to a heat transfer unitoperation 116. In at least some instances, control subsystem 290 maycontrol the flow rate or temperature of the cooling media passed throughthe reservoirs or coils that are thermally conductively coupled to loopreactor 101 or a conduit which has diverted a portion of the multi-phasemixture out of loop reactor 101 to a heat transfer unit operation 116.In other instances, the heat produced by the microbiological species maybe insufficient to maintain the multi-phase mixture in loop reactor 101within a desired temperature range. Such may occur, for example, inextremely cold environments where loop reactor 101 is located in anexposed or partially exposed exterior location. In some instances, thereservoirs or coils thermally conductively coupled to loop reactor 101or the conduit which has diverted portion of the multi-phase mixture outof loop reactor 101 to a heat transfer unit operation 116 may be used towarm the multi-phase mixture. In at least some instances, controlsubsystem 290 may control the flow rate or temperature of the warmingmedia passed through the reservoirs or coils 140 that are thermallyconductively coupled to the loop reactor 101 or the conduit which hasdiverted a portion of the multi-phase mixture out of the loop reactor101 to a heat transfer unit operation 116.

At 510, the pressure on the gas substrate bubbles traveling with themulti-phase mixture through loop reactor 101 is decreased by flowing themulti-phase mixture through a first pressure reduction device. In someinstances, the pressure on the gas substrate bubbles is decreased byflowing the multi-phase mixture through a first pressure reductiondevice that does not rely upon differences in hydrostatic pressure tocause a reduction in pressure. In other words, in some instances, thepressure on the gas substrate bubbles traveling with the multi-phasemixture through loop reactor 101 is decreased without a substantialchange in the elevation of the centerline of the loop reactor 101 at theexit of the first pressure reduction zone 108 relative to the elevationof the centerline of the loop reactor 101 at the entrance to the firstpressure reduction zone 108. The pressure decrease at 510 can, in someinstances, advantageously increase the rate at which gas substratebubbles and other gases desorb from the multi-phase mixture.

At 512, the multi-phase mixture exits first pressure reduction zone 108and flows to the gas/liquid separation vessel 102. Gaseous material thathas desorbed from the multi-phase mixture can also flow to thegas/liquid separation vessel 102 along with the multi-phase mixture. Themulti-phase mixture entering the gas/liquid separation vessel 102 caninclude, but is not limited to the liquid containing unabsorbednutrients, microorganisms and gas substrate bubbles containingundissolved and unabsorbed gas substrate. Gases and liquid enteringgas/liquid separation vessel 102 separate into a gas phase and a liquidphase within gas/liquid separation vessel 102. Gases can be collectedfrom the headspace of gas/liquid separation vessel 102 while liquid canbe removed from the bottom of gas/liquid separation vessel 102. Inaddition to liquid, microorganisms can also be collected in gas/liquidseparation vessel 102 and removed from the bottom thereof. The liquidand microorganisms removed from the bottom of gas/liquid separationvessel 102 can be delivered to the inlet 129 of fluid flow unitoperation 104 for recirculation through loop reactor 101. In at leastsome instances, at least a portion of the collected gas may besubsequently processed or separated. At least a portion of the collectedgas may be recycled to the loop reactor as a gas substrate. In someinstances, at least a portion of the collected gas may be sold orotherwise disposed of. In at least some instances, at least a portion ofthe collected gas may be sold or traded as a fungible commodity. In atleast some instances, the collected gas may include one or more C₂₊hydrocarbon gases and compounds based thereupon having value as either afinished product or as a raw material in a subsequent process. In someinstances, the reactor is used to produce natural or non-naturalproducts, such as ethanol, acetate, butanol, isoprene, propylene,farnesene, enzymes, or other metabolites or cellular products whereinthe product is derived from a microorganism. In such cases, the productsmay be present in either the gas effluent 123 or the liquid effluent 125depending on the physical properties of the product.

In at least some instances, at least a portion of the collected liquidmay be subsequently processed or separated. For example, at least aportion of the liquid separated from the multi-phase mixture, which mayor may not include biosolids, can be recycled through loop reactor 101.For example, at least a portion of the separated liquid containingbiosolids may be combined with additional liquids and flowed through theloop reactor 101. Such recycle may advantageously provide an ongoing,continuous or semi-continuous, inoculation of the loop reactor 101 withestablished biological species. In some instances, at least a portion ofthe separated liquid may be collected and sold or otherwise disposed of.In at least some instances, at least a portion of the separated liquidmay be sold or traded as a fungible commodity. In at least someinstances, the separated liquid may include one or more C₂₊ hydrocarbonliquids, including but not limited to one or more alcohols, glycols, orketones.

At 514, microorganisms from gas/liquid separation vessel 102 can beremoved upstream of fluid flow unit operation 104 or downstream of fluidflow unit operation 104, for example, at biomass removal port 128. Thecollected microorganisms can be further processed to recover desiredproducts. In some instances, the microorganisms collected via biomassremoval port 128 can be introduced to a separation subsystem 250 forprocessing and recovery of desired products.

FIG. 6 shows a high level method for stimulating production of biomass600 that utilizes a system 100 including one or more loop reactors 101described in detail above with regard to FIGS. 2-4. The example biomassproduction method 600 uses identical or nearly identical steps to thosedescribed in detail with regard to the method for stimulating productionof biomass method 500 discussed in detail with reference to FIG. 5, withthe exception that the method for stimulating the production of biomassmethod 600 includes a step of reducing the pressure on the gas bubbleswithin the multi-phase mixture in the loop reactor by passing themulti-phase mixture through a second pressure reduction zone. Thedescriptions of steps 502, 504, 506, 508 and 510 in FIG. 5 apply tosteps 602, 604, 606, 608 and 610 of FIG. 6, respectively. Thedescription of step 514 of FIG. 5 applies to step 616 of FIG. 6.

At 612 in FIG. 6, the pressure on the gas substrate bubbles travelingwith the multi-phase mixture through loop reactor 101 is decreased byflowing the multi-phase mixture from the first pressure reduction zone108 to a second pressure reduction zone 112. In some instances, at 612,the pressure on the gas substrate bubbles is decreased by flowing themulti-phase mixture through a second pressure reduction device that doesnot rely upon differences in hydrostatic pressure to cause a reductionin pressure. In other words, in some instances, at 612, the pressure onthe gas substrate bubbles traveling with the multi-phase mixture throughloop reactor 101 is decreased without a substantial change in theelevation of the centerline of the loop reactor 101 at the exit of thesecond pressure reduction zone 112 relative to the elevation of thecenterline of the loop reactor 101 at the entrance to the secondpressure reduction zone 112. In other instances, at 612, the pressure onthe gas substrate bubbles is decreased by flowing the multi-phasemixture through a second pressure reduction zone 108 that does rely upondifferences in hydrostatic pressure to cause a reduction in pressure. Inother words, in some instances, at 612, the pressure on the gassubstrate bubbles traveling with the multi-phase mixture through loopreactor 101 is decreased by causing a change in the elevation of thecenterline of the loop reactor 101 at the exit of the second pressurereduction zone 112 relative to the elevation of the centerline of theloop reactor 101 at the entrance to the second pressure reduction zone112. In some instances, when pressure on the gas substrate bubbles isreduced at both steps 610 and 612, the magnitude of the pressuredecrease at 612 can be less compared to the magnitude of the pressuredecrease at 610. In some instances, these decreases in pressureadvantageously increase the rate at which gas substrate bubbles andother gases desorb from the multi-phase mixture.

At 614, the multi-phase mixture from first pressure reduction zone 108which has entered second pressure reduction zone 112 or 113 exits secondpressure reduction zone 112 or 113 and flows to the gas/liquidseparation vessel 102. Gaseous material that has desorbed from themulti-phase mixture can also flow to the gas/liquid separation vessel102 along with the multi-phase mixture. The multi-phase mixture enteringthe gas/liquid separation vessel 102 can include, but is not limited tothe liquid containing unabsorbed nutrients, microorganisms and gassubstrate bubbles containing undissolved and unabsorbed gas substrate.Gases and liquid entering gas/liquid separation vessel 102 separate intoa gas phase and a liquid phase within gas/liquid separation vessel 102.Gases can be collected from the headspace of gas/liquid separationvessel 102 while liquid can be removed from the bottom of gas/liquidseparation vessel 102. In addition to liquid, microorganisms can also becollected in gas/liquid separation vessel 102 and removed from thebottom thereof. The liquid and microorganisms removed from the bottom ofgas/liquid separation vessel 102 can be delivered to the inlet 129 offluid flow unit operation 104 for recirculation through loop reactor101. In at least some instances, at least a portion of the collected gasmay be subsequently processed or separated. At least a portion of thecollected gas may be recycled to the loop reactor as a gas substrate. Insome instances, at least a portion of the collected gas may be sold orotherwise disposed of. In at least some instances, at least a portion ofthe collected gas may be sold or traded as a fungible commodity. In atleast some instances, the collected gas may include one or more C₂₊hydrocarbon gases and compounds based thereupon having value as either afinished product or as a raw material in a subsequent process. In someinstances, the reactor is used to produce natural or non-naturalproducts, such as ethanol, acetate, butanol, isoprene, propylene,farnesene, enzymes, or other metabolites or cellular products whereinthe product is derived from a microorganism. In such cases, the productsmay be present in either the gas effluent 123 or the liquid effluent 125depending on the physical properties of the product.

In at least some instances, at least a portion of the collected liquidmay be subsequently processed or separated. For example, at least aportion of the liquid separated from the multi-phase mixture, which mayor may not include biosolids, can be recycled through loop reactor 101.For example, at least a portion of the separated liquid containingbiosolids may be combined with additional liquids and flowed through theloop reactor 101. Such recycle may advantageously provide an ongoing,continuous or semi-continuous, inoculation of the loop reactor 101 withestablished biological species. In some instances, at least a portion ofthe separated liquid may be collected and sold or otherwise disposed of.In at least some instances, at least a portion of the separated liquidmay be sold or traded as a fungible commodity. In at least someinstances, the separated liquid may include one or more C₂₊ hydrocarbonliquids, including but not limited to one or more alcohols, glycols, orketones.

Example

A microbial culture including Methylococcus capsulatus Bath co-culturedwith a small amount of C₂ and C₃₊ metabolizing microorganisms wereprocessed in a system for stimulating the production of biomass thatincludes a loop reactor in accordance with embodiments described herein.The loop reactor included a non-vertical pressure reduction zone thatincluded an adjustable flow control device in the form of a backpressure control valve. The flow rate and/or the pressure within theloop section of the reactor was controllable by opening or closing thevalve. The loop reactor also included a desorption gas inlet between thegas/liquid separation vessel and the adjustable flow control device. Theloop section of the loop reactor included five inlets for introducingoxygen gas and methane gas into the loop section. Two inlets fornitrogen gas and three inlets for ammonium hydroxide were present in theloop section downstream of the fluid flow unit operation and upstream ofthe adjustable flow control device. Inlets for acid, acid salt andalkali, such as sulphuric acid, phosphoric acid, sodium hydroxide,potassium hydroxide, ferrous sulphate, calcium chloride, magnesium,potassium and trace elements were present between the gas/liquidseparation vessel and the pump. Two heat exchangers were utilized toprovide heat transfer to and from the multi-phase mixture in the loopsection as needed. The loop reactor was operated with the adjustableflow control device set for different flow rates through the adjustableflow control device. Steady state conditions, such as volumetric pumpoutput, temperature of multi-phase mixture, pressure within loop sectionbetween the pump outlet and the adjustable flow control device,dissolved oxygen content of multi-phase mixture, oxygen volumetric flowrate into the loop section, volumetric flow rate of methane into theloop section, volumetric flow rate of nitrogen into the loop section,and/or pH of multi-phase mixture within the loop reactor varieddepending upon the degree to which the control valve was open. With theflow rate through the control valve set at a specific level and the loopreactor in steady state operation, the following conditions wereobserved. Temperature within the loop reactor was measured to be about45 degrees Celsius. pH of the multi-phase mixture at the inlet to thepump was about 6.2 pH of the multi-phase mixture at the inlet to theadjustable flow control device was about 5.3 and about 7.9 between thepump and the adjustable flow control device. Density of the multi-phasemixture was about 1.7 kg/m³ at the outlet of the pump. Dissolved oxygencontent varied from 0.07 to 0.36 ppm at different locations within theloop section. Pressure upstream of the pump was about 0.6-0.7 bar gauge.Pressure downstream of the pump was about 3.0 bar gauge. Pressure at theinlet to the adjustable flow control device was about 1.9 bar gauge andpressure within the headspace of the gas/liquid separation vessel wasabout 0.4 bar gauge.

The effect on biomass production rate in the loop reactor of increasingor decreasing the flow rate through the control valve and the pressurewithin the loop section of the reactor was evaluated. During steadystate operation of the loop reactor, the opening of the control valvewas varied so that the flow rate through the control valve and thepressure in the loop section of the loop reactor was increased ordecreased. After the flow rate through the control valve was changed,the loop reactor was allowed to settle into steady state operation.After the loop reactor settled into steady state operation, data wascollected to determine the loop reactor's production rate after the flowrate through the control valve and the pressure within the loop sectionwas changed. The following is a summary of the findings of thatevaluation.

Increasing the pressure within the loop section by reducing the flowrate through the control valve resulted in an increased biomassproduction rate in the loop reactor compared to the production ratebefore the flow rate through the control valve was decreased. Increasingthe pressure within the loop section by reducing the flow rate throughthe control valve also produced higher pressures in the loop sectionbetween the control valve and the outlet of the pump and lower pressuresin the loop section between the outlet of the control valve and thegas/liquid separation vessel. Decreasing the pressure within the loopsection by increasing the flow rate through the control valve resultedin a decreased biomass production rate in the loop reactor compared tothe production rate in the loop reactor before the flow rate through thecontrol valve was increased. Decreasing the pressure within the loopsection by increasing the flow rate through the control valve producedlower pressures in the loop section between the control valve and theoutlet of the pump and higher pressures in the loop section between theoutlet of the control valve and the gas/liquid separation vessel. Thisexample illustrates how systems for stimulating the production ofbiomass that include a loop reactor in accordance with embodimentsdescribed herein are able to adjust the rate at which biomass isproduced in a loop reactor.

The above description of illustrated embodiments, including what isdescribed in the Abstract, is not intended to be exhaustive or to limitthe embodiments to the precise forms disclosed. Although specificembodiments of and examples are described herein for illustrativepurposes, various equivalent modifications can be made without departingfrom the spirit and scope of the disclosure, as will be recognized bythose skilled in the relevant art. The teachings provided herein of thevarious embodiments can be applied to other systems for stimulating theproduction of biomass, fermentors and fermentation systems. Such systemsfor stimulating the production of biomass, fermentors and fermentationsystems may include loop reactors or fermentors for purposes other thanchemical intermediate production, and may include loop reactors,fermentors and fermentation systems useful in food or beverageproduction. Similarly, the ancillary systems described herein, includingthe cooling gas/liquid separation unit operation, fluid flow unitoperation, nutrient supply subsystem, heat transfer unit operation andthe control subsystem may include a single system, for example a packageheat exchanger or package control system, or may include a customdesigned subsystem including any number of subcomponents that arephysically, fluidly, and communicably coupled in a manner facilitatingthe controlled production and distribution of cooling or warming media(i.e., by the heat transfer unit operation), facilitating the separationof at least a portion of the multi-phase mixture into a gas, liquid, andsemi-solid for recycle or for recovery and subsequent processing or sale(i.e., by the gas/liquid separation unit operation). The controlsubsystem can include an integrated or distributed control system thatprovides monitoring, alarming, control, and control output for all or aportion of the biomass production system or any of the ancillarysubsystems. The control subsystem may also include any number ofindividual loop controllers and the like for control of one or moreaspects of the biomass production system or any of the ancillarysubsystems.

The foregoing detailed description has set forth various embodiments ofthe devices and/or processes via the use of process flow diagrams andexample methods. Insofar as such block diagrams, schematics, andexamples contain one or more functions and/or operations, it will beunderstood by those skilled in the art that each function and/oroperation within such block diagrams, flowcharts, or examples can beimplemented, individually and/or collectively, using wide range ofoff-the-shelf or customized components that are well known to those ofskill in the chemical engineering arts. The microbiological specieslisted herein are intended to provide a sample of the potentialmicrobiological species that can be supported in a system for promotingthe production of biomass and loop reactors as described herein.

The various embodiments described above can be combined to providefurther embodiments. These and other changes can be made to theembodiments in light of the above-detailed description. In general, inthe following claims, the terms used should not be construed to limitthe claims to the specific embodiments disclosed in the specificationand the claims, but should be construed to include all possibleembodiments along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

The invention claimed is:
 1. A system for stimulating production ofbiomass comprising: a loop reactor, the loop reactor including: agas/liquid separation vessel for separating a multi-phase mixture of agas and a liquid culture medium into a gas phase and a liquid phase, thegas/liquid separation vessel including an outlet and an inlet; a loopsection including an inlet in fluid communication with the outlet of thegas/liquid separation vessel and an outlet in fluid communication withthe inlet of the gas/liquid separation vessel, the loop sectionincluding a loop section centerline and sloped upward between the inletof the loop section and the outlet of the loop section; and a firstnon-vertical pressure reduction zone including a first pressurereduction device, the first non-vertical pressure reduction zone locatedbetween the inlet of the loop section and the outlet of the loopsection, wherein a vertical distance between the loop section centerlineat the inlet of the gas/liquid separation vessel and the loop sectioncenterline at the inlet of the loop section is less than 8 meters. 2.The system of claim 1, wherein the first pressure reduction device isselected from a flow control device and an expansion joint.
 3. Thesystem of claim 1, further comprising a second pressure reduction zonedownstream of the first non-vertical pressure reduction zone.
 4. Thesystem of claim 3, wherein the second pressure reduction zone is asecond non-vertical pressure reduction zone.
 5. The system of claim 1,wherein the vertical distance between the loop section centerline at theinlet of the gas/liquid separation vessel and the loop sectioncenterline at the inlet of the loop section is less than 6 meters. 6.The system of claim 1, wherein the vertical distance between the loopsection centerline at the inlet of the gas/liquid separation vessel andthe loop section centerline at the inlet of the loop section is lessthan 5 meters.
 7. The system of claim 1, wherein the loop reactorfurther comprises a desorption gas inlet, the desorption gas inletlocated in a non-vertical portion of the loop section of the loopreactor.
 8. The system of claim 1, wherein the first pressure reductiondevice is a device that reduces pressure without relying upon a changein hydrostatic pressure.
 9. The system of claim 1, wherein the inlet ofthe gas/liquid separation vessel is spaced vertically from the outlet ofthe gas/liquid separation vessel.
 10. The system of claim 1, wherein thefirst pressure reduction device of the first non-vertical pressurereduction zone comprises a variable pressure reduction device.
 11. Thesystem of claim 1, wherein the loop section includes a downward slopingsection.
 12. The system of claim 1, wherein the loop reactor furthercomprises a desorption gas inlet upstream of the first non-verticalpressure reduction zone.
 13. The system of claim 1, wherein the inlet ofthe gas/liquid separation vessel is above the outlet of the gas/liquidseparation vessel.
 14. A system for stimulating production of biomasscomprising: a loop reactor, the loop reactor including: a gas/liquidseparation vessel for separating a multi-phase mixture of a gas and aliquid culture medium into a gas phase and a liquid phase, thegas/liquid separation vessel including an outlet and an inlet; a loopsection including an inlet in fluid communication with the outlet of thegas/liquid separation vessel and an outlet in fluid communication withthe inlet of the gas/liquid separation vessel, the loop sectionincluding a loop section centerline; and a first non-vertical pressurereduction zone including a first pressure reduction device including aneccentric reducer, the first non-vertical pressure reduction zonelocated between the inlet of the loop section and the outlet of the loopsection, wherein a vertical distance between the loop section centerlineat the inlet of the gas/liquid separation vessel and the loop sectioncenterline at the inlet of the loop section is less than 8 meters. 15.The system of claim 14, wherein the first pressure reduction devicefurther comprises an eccentric expander downstream from the eccentricreducer.
 16. The system of claim 15, wherein the first pressurereduction device further comprises a control valve between the eccentricreducer and the eccentric expander.
 17. The system of claim 14, whereinthe loop section includes a portion that slopes upward.
 18. The systemof claim 14, wherein the loop section includes a portion that slopesdownward.
 19. A system for stimulating production of biomass comprising:a loop reactor, the loop reactor including: a gas/liquid separationvessel for separating a multi-phase mixture of a gas and a liquidculture medium into a gas phase and a liquid phase, the gas/liquidseparation vessel including an outlet and an inlet; a loop sectionincluding an inlet in fluid communication with the outlet of thegas/liquid separation vessel and an outlet in fluid communication withthe inlet of the gas/liquid separation vessel, the inlet of the gasseparation vessel spaced above the outlet of the gas/liquid separationvessel, the loop section including a loop section centerline and aportion that slopes upward in a direction extending from the loopsection inlet to the loop section outlet; and a first non-verticalpressure reduction zone including a control valve and an eccentricexpander, the first non-vertical pressure reduction zone located betweenthe inlet of the loop section and the outlet of the loop section,wherein a vertical distance between the loop section centerline at theinlet of the gas/liquid separation vessel and the loop sectioncenterline at the inlet of the loop section is less than 8 meters. 20.The system of claim 19, further comprising an eccentric reducer upstreamof the eccentric expander.
 21. The system of claim 19, wherein the loopsection includes a portion that slopes downward.